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Research Article
Linking Engineered Cells to Their Digital Twins: A Version Control System for Strain EngineeringClick to copy article linkArticle link copied!
- Jonathan Tellechea-LuzardoJonathan Tellechea-LuzardoInterdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.More by Jonathan Tellechea-Luzardo
- Charles WinterhalterCharles WinterhalterInterdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.More by Charles Winterhalter
- Paweł WideraPaweł WideraInterdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.More by Paweł Widera
- Jerzy KozyraJerzy KozyraInterdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.More by Jerzy Kozyra
- Víctor de LorenzoVíctor de LorenzoSystems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, SpainMore by Víctor de Lorenzo
- Natalio Krasnogor*Natalio Krasnogor*Email: [email protected]Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.More by Natalio Krasnogor
Abstract
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As DNA sequencing and synthesis become cheaper and more easily accessible, the scale and complexity of biological engineering projects is set to grow. Yet, although there is an accelerating convergence between biotechnology and digital technology, a deficit in software and laboratory techniques diminishes the ability to make biotechnology more agile, reproducible, and transparent while, at the same time, limiting the security and safety of synthetic biology constructs. To partially address some of these problems, this paper presents an approach for physically linking engineered cells to their digital footprint—we called it digital twinning. This enables the tracking of the entire engineering history of a cell line in a specialized version control system for collaborative strain engineering via simple barcoding protocols.
Copyright © 2020 American Chemical Society
There is a rapidly accelerating convergence between biotechnology and information and communication technologies (ICT).
As writing and reading DNA becomes routine, cheaper, and pervasive, genetic engineering—that is, programming biological organisms—becomes more similar to programming computers. This has important disruptive implications for biotechnology: (1) larger teams of bioprogrammers—both in biotechnology companies of all sizes and in research organizations such as universities—work together and concurrently in the genetic programming of biological organisms; (2) the pace of innovation for biological “apps” is set to explode; and (3) the ecosystem and supply chain of biotechnological products, in particular cell lines and plasmids, is to become more complex and more diversified.
In software development, the time to market for new products has shortened dramatically over the recent years, thanks to (among other factors) advances in continuous integration and deployment allowing teams of programmers (often geographically distributed), to rapidly develop, test, debug, and promptly push new code to production. This means that new software products can reach their users and customers faster than ever before.
The core element of the technology underpinning continuous integration and deployment are the Version Control Systems (VCS). Their roots date back to Bell Laboratories’ early 1970s Source Code Control System. (1) Git, the prevalent VCS powering modern software engineering, was introduced by Linus Torvalds in 2005 and popularized the use of distributed version control systems.
Nowadays distributed VCS are used daily by software developers across the world. VCS help distributed teams to keep track of the history of changes to large software systems as it enables them to quickly answer questions such as “What was done to a piece of software?”, “What versions of the code are available?”, “Who introduced the last feature (or bug) into the code?”, “When did the modification take place?”, “What was modified?”, “How does this new version differ from the previous one?”, and importantly “Why was this piece of software modified?”, etc.
A version control system provides answers to the above questions, greatly simplifying the work of distributed teams of programmers who must modify the same piece of software concurrently. VCS achieve this by storing the files in the repository together with the entire history of changes to them; changes are automatically tracked and semiautomatically merged by the system. Because the entire lineage of a source code is maintained, VCS enables traceability, transparency, and backtracking (if necessary) as well as branching out new versions of computer code. Branched code does not interfere with the original code and yet retains all the metadata that lead to that branch’s origins, thus providing a safe sandpit for experimentation (i.e., if something breaks, one can always restore previous states). Without VCSs it would be virtually impossible to develop complex computer programs as we know them today. For a recent biology and biotechnology friendly introduction to version control and Git, refer to ref (2).
How would a biotechnology-specific version control system contribute to making biotechnology more agile, reproducible, and transparent—and the corresponding live constructs more traceable for the sake of their security and safety? (3)
On one hand, mislabeling and misidentification of biological samples occurs frequently in the laboratory. (4) While being able to properly identify and track a strain’s origins is essential, and notwithstanding persistent calls to address this challenge, (5−7) it is a problem that lacks appropriate tooling. Moreover, a related major problem affecting many scientific disciplines—including biotechnology—is experimental irreproducibility. (8) As recently as 2018, Nature had a special issue themed “Challenges in Irreproducible Research” to highlight some of the most urgent issues and suggest potential ways forward, with Brazil the first country—to the best of our knowledge—to launch a reproducibility initiative within its borders. (9)
Laboratory data generated by researchers also face problems like data loss and lack of accepted engineering and reporting standards. Electronic Lab Notebooks and other online applications that use the cloud to store and publish the data and experiments generated are a partial step in this direction, although they are generic for lab operations rather than specific to the genetic programming of organisms. Thus, new tools and software tools have been called for. (10)
On the other hand, although several methods aimed at identifying cell lines coming from clinical or environmental samples have been developed, little effort has been put into establishing such tools for laboratory-created strains. From an engineering viewpoint, one needs to know not only a strain’s genome sequence but, importantly, also the history of changes that were made to a strain (e.g., added plasmids, scars left by knock-ins/knockouts, etc.). Ideally, one would want to also maintain a detailed history of experimental conditions used, the intention behind the modifications done to a strain or to its growth media, as well as other metadata such as, e.g., the lab of provenance, the genetic engineer(s) who worked on a strain, etc.
The lack of robust, yet simple, solutions to address the issues mentioned above, on the one hand, leads to wasteful use of both public and private funds and, on the other hand, contributes to the growth in public mistrust due to a lack of traceability and transparency in biotechnology product innovation.
Moreover, current practice for linking a strain digital footprint to the actual biological sample is based around hand-written notes, word processor or spreadsheet files, and—in the best case—the labeling of test tubes with barcodes generated from generic laboratory information management systems. Crucially, in all those cases, the biological sample itself does not carry a record of its digital footprint. That is, with current laboratory practice, a strain’s digital footprint (e.g., strain designs, genome sequence, recombineering sites, engineering history, etc.) is disconnected from the physical strain sample: no actual connection exists between the strains one creates in the laboratory (or that is used in production, e.g., to manufacture enzymes or biologics at scale) and the data available for that strain. Recent advances in DNA synthesis techniques and information storage in DNA could help bridge this important gap.
The usage of DNA as a long-term way of storing data has been recently demonstrated. (11) The use of small, synthetic DNA sequences as identifiers has been reported several times in very diverse areas. These short DNA sequences allow the identification of mutants in mixed populations in both microbial studies (12,13) and tumor cell lines studies under different treatments. (14,15) Also, DNA barcodes have been used to track cell lineage by progressively editing the barcode sequence using CRISPR. (16) DNA barcodes can be used in gene synthesis, in which the barcode sequence is used to isolate and assemble a synthetic gene (17) or large pathways in various circuit designs. (18) Additionally, DNA barcodes have been shown to be useful in drug discovery by tagging and identifying several chemicals that bind to specific target molecules. (19) Very recently new barcode generation algorithms have been developed to consider any type of synthesis or sequencing mistake which increases the robustness of all the previous uses. (20)
Finally, a version control system that physically links, via an embedded DNA barcode, engineered cells to their digital twins would also contribute toward more responsible research and innovation. For example, the efficacy of genetic firewalls, sophisticated as they might be, may not be able to avoid engineered organisms to escape a given niche. (22) Existing proposals for containing genetically programmed organisms and SynBio agents, do not seem to go beyond events occurring at frequencies of 10–11, which is not enough for what has been called certainty of containment (CoC). (21) In contrast, most of the concerns on undesirable propagation of human-made constructs could be managed if chassis and agents were barcoded with specific and unique DNA sequences that—once decoded—could trace back the sample to its origins. Barcoded clones would thus be equivalent to pets implanted subcutaneously with identification chips: in case they get lost or do some harm, their owner and their lineage can be immediately identified, and measures taken for counteracting detrimental effects. By the same token, barcoded strains would allow accessing all relevant information on its pedigree, safety and modifications implemented in them. Moreover, it would allow for a detailed comparison of the current genome sequence at the time of sampling and the intended (initial) genomic sequence at the moment of engineering. This would certainly facilitate comparative genomics and evolutionary studies starting from a well characterized point of origin for the later collected (i.e., escaped) samples. Barcoding would thus be complementary to—if not even more practical than—containment measures which as mentioned above are unlikely to deliver CoC where a very large cell population is at stake. Instead, barcodes will not only make traceability simple, but it will also assign a nonambiguous cipher to the growingly improved versions of the same chassis (as is the case with computers and mobile phones operating systems).
In this paper we present a biotechnology specific version control system, CellRepo, that provides both the genetic toolkits and web-based software to physically link living samples to their digital footprint history. CellRepo is based on small, unique and bio-orthogonal DNA sequences inserted in specific genomic locations of a strain. By a single sequencing reaction, the DNA sequence can be retrieved, and hence the user can track down the entire digital footprint history of a strain via our web server: strain creators, parental and derivative strains, strain design documentation, related papers, experimental protocols, computer models, etc. can all be retrieved from the proposed system (Figure 1). Put together, the biotechnology kits and the software repository move the digitalization of biotechnology a step closer making it more collaborative, scalable, transparent, traceable, and reproducible.
Figure 1
Figure 1. An example of version control in strain engineering. A repository with the master culture (red) is branched by two different biologists to create new Strain 1 (purple) and Strain 3 (green). Each dot in the commit tree represents a strain engineering milestone and is created by modifying the parent strand (or an earlier derivative). For critical (user defined) steps in the engineering process, a unique identifier can be generated and stored as a DNA barcode in the strain chromosome. The barcode is associated with a “commit” within our version control system, with each commit containing the digital footprint (files and other metadata information) that lead to this milestone.
Materials and Methods
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We report next the materials and methods used for physically relating a given strain to its digital twin within in the version control system. This is accomplished via writing and reading of a unique DNA barcode (Figure 2) that is demonstrated with different barcoding techniques in the model organisms Escherichia coli and Bacillus subtilis. When a barcoded strain is modified and a new milestone is reached, the strain can be rebarcoded: the new barcode replaces the previous one (Figure 2 Bottom). No information is encoded in the barcode itself, only the reference to the web profile. Updating a barcode only requires replacing one by another without the need for concatenation. Barcode design details are available in the Supporting Information.
Figure 2
Figure 2. Writing a barcode (Top): During strain engineering, a key milestone is reached, and the strain is barcoded with a unique DNA sequence generated by the CellRepo website. A commit to the repository is made connecting the unique barcode (now in the chromosome of the cell) with all the cell’s related documentation. Reading a barcode (Center): A DNA barcode is read from a sample strain enabling the lookup of all the strains’ documentation from the CellRepo repository. Updating a barcode (Bottom): Strain 1 was barcoded with Barcode-1. When the strain is modified to give Strain 2 a new barcode must be used. The strain needs to be updated with Barcode-2 replacing Barcode-1. The repository keeps track of the barcode’s “lineage”; thus, via sequencing Barcode-2 one can trace back the parental strain (and its own barcode).
Materials
DNA was amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs, NEB). For cloning purposes DNA was purified using Monarch PCR & DNA Cleanup Kit (NEB) and assembled using NEBuilder HiFi DNA Assembly Cloning Kit (NEB). Plasmid preparations were carried out using QIAprep Spin Miniprep Kit (QIAGEN). Primers and Synthetic DNA sequences (barcode gBlocks) were synthesized by Integrated DNA Technologies. All barcodes were sequenced by Sanger sequencing via a universal sequencing primer.
Strains and Plasmids
E. coli DH5α cells were used for most of plasmid preparations. Plasmids carrying a R6K-γ origin of replication were prepared and stored using DH5α/λ-pir cells. We used BW25113 as the barcode receiver E. coli strain and used its genomic DNA as template for homologous recombination to insert barcodes on the chromosome. Bacillus subtilis experiments were conducted in 168CA strain for barcode insertion, and the toxin/antitoxin unit for barcode selection/counter-selection was amplified from B. subtilis ZPM6. (23) All the “vector” tagged plasmids were built containing a restriction site that allows easy barcode sequence cloning. See Table 1 for a full list and description of the plasmids used in this work. Plasmid maps can be found in Figure S3 in the Supporting Information.
Table 1. Plasmids Used in Our Barcoding Kitsa
| species | task | plasmid name | antibiotic resistance | features | ref. |
|---|---|---|---|---|---|
| E. coli | λ-red | pKD46 | Ampicillin | λ-red genes under pBad promoter, temperature sensitive | (24) |
| pCP20 | Ampicillin | FLP recombinase gene, temperature sensitive | (24) | ||
| pEC-Vector | Ampicillin, Chloramphenicol | CmR gene between FRT sites, homologous arms, R6k-γ origin. BamHI | This study | ||
| pEC-BC | Ampicillin, Chloramphenicol | Barcoded pEC-vector | This study | ||
| CRISPR | pREDCas9 | Spectinomycin | Constitutive Cas9 expression, λ-red genes under IPTG inducible promoter, sgRNA targeting pUC origin under pBAD promoter, temperature sensitive replicon | (25) | |
| pEC-CRISPR-vector | Ampicillin | pUC origin, gRNA and homologous arms targeting barcoding region. SphI | This study | ||
| pEC-CRISPR-BC | Ampicillin | Barcoded pEC-CRISPR | This study | ||
| B. subtilis | Cre-Lox | pBS-CreLox-Vector | Spectinomycin, Zeocin | ZeoR gene between loxP sites and homologous arms. SpeI | This study |
| pBS-CreLox-BC | Spectinomycin, Zeocin | Barcoded version of pBS-CreLox-Vector | This study | ||
| pDR244 | Ampicillin | Cre recombinase expression, temperature sensitive | (26) | ||
| CRISPR | pJOE8999.1 | Kanamycin | Cas9 under mannose inducible promoter, sgRNA under constitutive promoter, temperature sensitive | (27) | |
| pBS-CRISPR-Vector | Kanamycin | From pJOE8999.1. Homologous arms, sgRNA targeting barcoding location. SpeI | This study | ||
| pBS-CRISPR-BC | Kanamycin | Barcoded pBS-CRISPR-Vector | This study | ||
| GFP insertion | pGFP-rrnB | Ampicillin, Chloramphenicol | GFP gene under constitutive promoter and CmR gene in between homologous arms targeting amyE locus. Nonreplicative in B. subtilis. | (28) |
a
Restriction sites used to clone the barcode are shown in italics. pREDCas9 was a gift from Tao Chen (Addgene plasmid # 71541).
Barcoding Site Selection
Barcodes irrevocably link a strain to its digital twin and should be inserted on the chromosome in a favorable genetic context. To maximize sequence stability and to minimize genetic interference with a strain’s phenotype, we chose to insert the barcodes far from important gene loci. On the chromosome, interacting regulatory units are often found in neighboring locations. Not all genes in the genome are essential, and therefore, for each species, we gathered and curated data about essential genes from the literature and ruled out locations in the genome that were within the operon of essential genes. (29,30) Genes involved in metabolism regulation, cell wall components and migrating elements were also avoided. Following these considerations, barcodes were inserted at specific loci that did not affect replication upon cell division and, at the same time, were placed in a favorable biological context. All in all, the barcodes inserted at these loci (Figure 3) are unlikely to interact with proximal elements or to interfere with strain physiology.
Figure 3
Figure 3. Graphic representation of the barcoding sites for both species. Orange lines show homologous regions used for barcoding purposes. Coordinates indicate start and end of nearby genes. Green blocks correspond to universal primer binding sites (25 bp). Yellow and red blocks represent synchronization (9 bp) and checksum sequences (18 bp) respectively (Supporting Information). For E. coli and B. subtilis, NCBI genome Accession number CP009273.1 and AL009126.3 were used, respectively.
E. coli Barcoding
For detailed barcoding protocols, please see the Supporting Information.
λ-Red Mediated Recombination
We assembled pEC-vector containing the R6k-γ origin of replication and a selection cassette (cat gene, conferring chloramphenicol resistance) flanked by homology arms for genomic insertion (Figure 3) and FRT (Flipase Recognition Target) sequences for antibiotic cassette removal. We subsequently inserted barcode sequences on this parental plasmid leading to pEC-BC and adapted the protocol described by (24) for chromosomal insertion. Cells were first transformed with the helper plasmid pKD46 for expression of λ-red enzymes that allow barcode insertion via double crossover. Transformant colonies were selected in the presence of carbenicillin (100 μg/mL) at 30 °C. Carbenicillin resistant colony was inoculated and induced with l-arabinose 30 mM. When OD = 0.5, cells were made electrocompetent.
We amplified the barcode recombinant assembly from pEC-BC by PCR. Electrocompetent cells were mixed with the barcoding cassette. Cells were electroporated (25 μF, 200 Ω, 1.8 kV, Gene Pulser, BioRad) and plated in LB plates supplemented with chloramphenicol (25 μg/mL) at 37 °C. Barcoding was checked by colony-PCR. If selection cassette removal was required, barcoded colonies were transformed with the pCP20 helper plasmid and incubated at 30 °C in LB/Carbenicillin plates. Carbenicillin resistant colonies were grown at 37 °C in LB plates to promote the loss of pCP20. Carbenicillin and chloramphenicol sensitive clones were verified by colony PCR and positive clones were stored as barcoded strains.
CRISPR
E. coli cells were also barcoded by CRISPR using a two-plasmid system. (25,31) The plasmid pEC-CRISPR-vector was constructed by cloning the homologous arms and the 20N-gRNA scaffold under the control of a constitutive promoter. The barcode sequence was added afterward in between both homologous arms by HiFi DNA Assembly leading to the pEC-CRISPR-BC plasmid. pREDCas9 was transformed into E. coli BW25113 cells and selected in LB/Spectinomycin (50 μg/mL) at 30 °C. λ-Red recombinase was induced with IPTG 2 mM until OD = 0.5. pEC-CRISPR-BC was transformed and selected in LB/Spectinomycin/Carbenicillin plates. Transformant cells were checked by colony PCR. Positive clones were cured from pEC-CRISPR-BC by l-arabinose induction (30 mM). pREDCas9 was cured afterward by growing cells at 37 °C. Spectinomycin and carbenicillin sensitive clones were stored.
B. subtilis Barcoding
For detailed barcoding protocols, please see the Supporting Information.
Toxin/Antitoxin
We assembled a recombinant cassette containing homology arms, the mazF-ZeoR unit for selection/counter-selection and barcode sequence by SOE-PCR and adapted the protocol described in ref (23) to promote chromosomal insertion. The PCR product was transformed into B. subtilis cells. Cells were plated in NA (Nutrient agar)/zeocin(20 μg/mL) plates. Colonies were restreaked on NA/zeocin plates and tested for the integration of the recombinant DNA by PCR. For mazF-ZeoR cassette removal, positive clones were grown with 1% xylose to induce the toxin gene. Cells were plated in xylose supplemented media. Individual colonies were restreaked on NA and NA/zeocin plates; colonies which tested positive by PCR and sequencing were stored as barcoded strains.
Cre-Lox
We adapted the Cre-Lox protocol from ref (26) and constructed a plasmid with zeocin antibiotic resistance flanked by loxP sites and homologous regions to the barcode insertion site. Barcodes were cloned into this parental plasmid, the recombinant cassette was PCR amplified and used to transform B. subtilis 168CA. Zeocin resistant colonies were transformed with pDR224 at 30 °C, and spectinomycin (100 μg/mL) resistant colonies were verified for zeocin cassette removal by colony-PCR. Positive colonies were cured from pDR224 after incubation at 37 °C. Spectinomycin and zeocin sensitive colonies were stored as barcoded strains.
CRISPR
To barcode B. subtilis cells using CRISPR we used a single-plasmid approach. (27) Homologous arms and sgRNA target sequence were cloned in pJOE backbone. The barcode DNA sequence was cloned in afterward. 168CA cells were transformed with this plasmid and selected in LB/Kanamycin (5 μg/mL) supplemented with 0.2% mannose for Cas9 induction at 30 °C. Transformants were checked by colony PCR. pBS-CRISPR-BC was cured by growing the cells at 37 °C. Kanamycin sensitive cells were stored as barcoded strains.
Refer to Supplementary Figure S3 for detailed plasmid maps for each method.
Barcode Sequence Retrieval
To easily check the integrity of the barcode sequences, all barcode identifiers were tagged with a Universal Primer sequence (5′-TGGACATACATAGTATACTCTGGTG-3′). This primer is used during Sanger sequencing to retrieve any barcode sequence. Also, it can be used to check the success of the barcoding experiment (through colony-PCR) together with appropriate species-specific reverse primers.
Barcode Stability Assay
Chemostat
Using a chemostat we followed bacteria over 200 generations (about 4 days of culture) and retrieved barcode information at regular time points (every 15–25 bacterial generations, i.e., passage through the entire cell cycle). For E. coli the same cultures were followed over 200 consecutive generations, while in B. subtilis, cells were followed for 100 generations, induced to stress and sporulation by ethanol treatment and regrown from spores for a further 100 generations. Barcode sequences were obtained by PCR product Sanger sequencing after barcode amplification from genomic DNA.
For this study, we performed CFU growth curves and serially diluted cultures over time to obtain ideal dilution rates at which a minimal number of cells could be used to inoculate a chemostat. Provided this minimum number of cells, it was possible to evaluate, given a specific growth rate, the time needed for cultures to attain exponential phase (e.g., when the chemostat continuous flow should be turned on). Along all chemostat experiments, we recorded optical density (OD600) measurements while sampling cells for barcode sequencing to ensure estimated dilution rates were accurate and cultures could reach steady-state growth (Figure S4 in Supporting Information).
Large-Scale Growth Assay
With automated plate handling systems, we compared the evolution of 384 subcultures of barcoded and control cells over several stationary phase redilutions, in order to observe any changes represented in growth defects. Besides growth characterization, all barcoded samples were sequenced to uncover any potential mutations in DNA barcode sequences.
We used a liquid handling robot (Beckman Coulter Biomek FX) and an automated plate reader to handle 8 individual 96-well plates simultaneously. Before starting a growth experiment, plates were sealed by a gas-permeable membrane. In this assay, we followed bacteria over 10 subculture experiments. Initially, a single colony from each barcoded/control strain was picked from a fresh plate and grown in 25 mL LB supplemented with 0.4% (w/v) glucose overnight at 37 °C with regular shaking parameters (about 150 rpm). In the morning, saturated cultures were spun down, resuspended in fresh LB medium, diluted 100 times and 200 μL were loaded onto ThermoFisher clear 96-well microplates.
For all subculture experiments, two conditions were tested: an early stop of bacterial cultures after 6 h (in late exponential/start of stationary phase) and a prolonged culture in stationary phase (12 h) before snap freezing. For the first subculture, 100 μL were harvested after 6 h for the early sampling point, and the remaining bacterial culture was further incubated up to 12 h. All subsequent cultures were diluted 100 times from frozen stocks and cultivated in a 100 μL total volume for both early and late sampling points (Figure S5 and Figure S6 of the Supporting Information). By the end of the 10 subcultures, genomic DNA (gDNA) was extracted and screened for potential variations in barcode sequences.
Results
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We illustrate all the concepts introduced by performing a simple genetic engineering experiment that includes barcoding a cell line and uploading to the version control system. B. subtilis 168CA wild-type strain was barcoded with Barcode 659 using the Cre-Lox method described before. The resulting strain was then transformed with pGFP-rrnB. (28) Chloramphenicol (5 μg/mL) resistant clones were checked by colony-PCR and by checking the green fluorescence emission in a plate reader. This new strain was rebarcoded using Barcode 207. Two repositories can be found in our webpage describing B. subtilis (https://cellrepo.ico2s.org/B.subtilis-GFP) and E. coli experiments (https://cellrepo.ico2s.org/E.coli-Barcoding).
Barcoding Process
All the methods used to barcode both species probed to be capable of barcoding the cells with high efficiency (Supplementary Table S1). After extracting the genomic DNA and PCR the barcode, it was always possible to retrieve the barcode DNA sequence.
Barcode Stability Assay
After the chemostat experiment, we analyzed 128 sequencing reactions after 200 generations, including 24 controls to compare the evolution of barcoded vs wild-type strains. We confirmed that control wild type sequences remained unchanged and found no variation in barcode sequences over 200 generations for either species. This is consistent with mutation rate bibliographic data in both species (32,33) (Supplementary Table S2).
In the large-scale growth assay, over 10 subcultures, we estimated from 100-fold dilutions of previous subcultures that final samples reached about 100 generations. In our assay, sequencing of 384 barcoded strains tested in a normal vs stress conditions always revealed intact DNA barcode sequences. No major difference in growth rates was observed across the different samples (Supplementary Figures S5 and S6). Most sequencing reads left a 26–27 nucleotide gap downstream of the universal primer-binding site (due to Sanger sequencing limitations) and then showed a perfect match with the expected alignment. In less than 5% of cases, sequencing data quality was noisy, but a second complementary read would always manage to recover the integrity of a barcode sequence. The screening of a large number of biological replicates demonstrated the robustness of the barcode insertion process as well as the barcode sequence stability over time.
Web Server
We implemented the software aspects of the CellRepo by extending existing technology—the source code management system Kallithea (https://kallithea-scm.org). The web server hosting our implementation is available at https://cellrepo.ico2s.org and is free for academic use. We documented on the CellRepo server the key milestones of the process of cell engineering and barcoding described earlier (Figure 4), so that the reader can access additional information directly there. The CellRepo server allows new users (upon registration) to create their own cell engineering repositories. After that, the users can add/modify files and generate barcodes (Figure 5 (c)). There is no limit on the type of data that can be associated with a repository. Data might include plasmid designs, FASTA or GenBank files with genomic or plasmid data, SBOL files, characterization of experiments outputs (e.g., optical density readouts, growth curves, experimental protocols, etc.), references to papers or the papers themselves, etc. (Figure 5 (b)). All changes/commits are organized in a chronological order. Every repository has a wiki-like front-page that describes the essential details (e.g., genotype, phenotype, owner, etc.) (Figure 5 (a)). Furthermore, the system allows a repository to be forked, so further work could be carried into a cell line without interfering with the original repository (Figure 5).
Figure 4
Figure 4. Screenshot of the repository summary page. The list of changes shows all steps made in the process of engineering a Bacillus subtilis mutant strain with inserted GFP gene and represents a digital footprint of the cell line. Two key engineering milestones (revision 4 and 10) have been linked via a genetic barcode to the cell line.
Figure 5
Figure 5. Examples of information stored in the repository: (a) essential strain details, (b) list of added files that are part of the digital documentation for a cell line at a given point in time, and (c) list of revisions barcoded into the cell line, called “snapshots”, as they uniquely link to the digital documentation (i.e., the digital twin) at a specific point in the cell engineering cycle. In this example revision with id r10:acc4afc32349 and r4:070c713ad448 have been barcoded back into the cell line.
Discussion
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In this paper we argue that as the speed, size, and complexity of synthetic biology, biotechnology, and genetic engineering approaches increase, new tools are required to handle the substantial scale up that is taking place. We propose a new purpose-built version control system, CellRepo, for strain engineering. CellRepo links biological cells to their “digital twins” thus allowing the tracking of all extant data and metadata related to a cell engineering process. This link is created by placing a barcode into the cell that can—at a later stage—be retrieved via a simple sequencing reaction. The retrieved barcode can then be used to identify the cell line and all the related details stored in CellRepo (who built the cell line, in which lab, when the modifications took place, what protocols were used, what was done to the cell and why, etc.).
A possible limitations to this new technology may include inconsistencies between the barcode (and its digitally stored information) and the strain itself: mutations in the strain’s genome may arise after the strain has been barcoded leading to a discrepancy between the genome sequence associated with a barcode and the genome of the mutated strain. Without the barcode and its associated digital footprint in the repository, this situation will not be detectable because it will not be possible to know what the original strain genome was. With a barcode, even if the strain changed, there is the possibility to link back to the intended strain, assess the speed of the “evolutionary clock” and ascertain whether the changes are tolerable or not (which is—of course—application specific).
In the examples in this paper, the barcoding sites were chosen after considering a simple case where the barcodes were inserted in a neutral spot in the genome of two species, which have a wide range of genetic tools available and well annotated genomes. Similar strategies to the ones used in this paper may be followed for other well annotated organisms. For less well-known organisms, the choice of barcoding loci is less clear and remains an open avenue for future research. However, a practical guide is that, when lacking clear “neutral” barcoding loci, the barcode might be inserted together with the genetic modifications that are being constructed. Provided that the position of the identifier in the genome is well-documented within the digital information on the strain, our approach should still equip genetic engineers with the means to properly barcode their nonmodel organisms and link them to our version control system.
The barcoding protocols showed in this paper are not directly applicable to large-scale genomic rearrangements. (34) However, this kind of experiments utilize their own barcoding strategies that can still be linked to our version control system if the positions of their barcodes—within the massively rearranged genomes—are provided as part of “commits” to CellRepo.
As the need of barcoding new species and the number of synthetic biology chassis increases, fresh genetic technologies need to be developed for stably inserting such sequences in the genome of target organisms across the tree of life. Methods such as CRISPR prime (35) look like promising enablers that could expand the reach of the version control system into nonmodel organisms and also make the barcoding of more standard chassis easier.
We envision that adoption of barcoding as a routine for standardized identification of genetically engineered organisms will ease approval, security, and safety of the corresponding modified agents for industrial and environmental uses to a degree far superior than the current propositions for genetic firewalls—which do not provide a certainty of containment. Besides the computational effort, such regulation-oriented and safety-oriented barcoding will also demand genome editing of strains deficient in recombination, a feature typically desirable for environmental safety of GMOs. To this end, a number of molecular tools, e.g., counterselectable TargeTrons, are being currently developed in our laboratories. (36) In the meantime, CellRepo is freely available for noncommercial use at https://cellrepo.ico2s.org.
Supporting Information
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.9b00400.
Supplementary text explains barcode design; Supplementary figures of plasmid maps used for barcoding and description of barcode stability assays results; Supplementary tables show the efficiency of the barcoding methods and the mutation rate comparison with bibliography (PDF)
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Author Information
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- Natalio Krasnogor - Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.;
http://orcid.org/0000-0002-2651-4320;
- Jonathan Tellechea-Luzardo - Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.;http://orcid.org/0000-0003-2198-4558
- Paweł Widera - Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, Newcastle Upon Tyne NE4 5TG, U.K.;http://orcid.org/0000-0003-4955-3653
- Víctor de Lorenzo - Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain;http://orcid.org/0000-0002-6041-2731
J.T.L., C.W., J.K., and N.K. were supported by the UK Engineering and Physical Research Council under project “Synthetic Portabolomics: Leading the way at the crossroads of the Digital and the Bio Economies (EP/N031962/1)”. N.K. is funded by a Royal Academy of Engineering Chair in Emerging Technology award. V.d.L. was supported by project “BioRoboost (H2020-NMBP-BIO-CSA-2018, grant agreement N820699)”.
References
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This article references 36 other publications.
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- 7Masters, J. R. (2012) End the Scandal of False Cell Lines. Nature (London, U. K.) 492, 186, DOI: 10.1038/492186aGoogle Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVamtb3E&md5=a53b24e02777644432f56d9210d9a223Cell-line authentication End the scandal of false cell linesMasters, John R.Nature (London, United Kingdom) (2012), 492 (7428), 186CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)There is no expanded citation for this reference.
- 8Freedman, L. P., Cockburn, I. M., and Simcoe, T. S. (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol. 13, e1002165, DOI: 10.1371/journal.pbio.1002165Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XptFahuro%253D&md5=603580a3ddf0d8c55a22926fbc3ac8b0The economics of reproducibility in preclinical researchFreedman, Leonard P.; Cockburn, Iain M.; Simcoe, Timothy S.PLoS Biology (2015), 13 (6), e1002165/1-e1002165/9CODEN: PBLIBG; ISSN:1545-7885. (Public Library of Science)Low reproducibility rates within life science research undermine cumulative knowledge prodn. and contribute to both delays and costs of therapeutic drug development. An anal. of past studies indicates that the cumulative (total) prevalence of irreproducible preclin. research exceeds 50%, resulting in approx. US$28,000,000,000 (US $28B)/yr spent on preclin. research that is not reproducible-in the United States alone. We outline a framework for solns. and a plan for long-term improvements in reproducibility rates that will help to accelerate the discovery of life-saving therapies and cures.
- 9De Oliveira Andrade, R. (2019) Brazil’s Science Faces Reproducibility Test. Nature (London, U. K.) 569, 318– 319, DOI: 10.1038/d41586-019-01485-zGoogle Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpslGgu7w%253D&md5=e2f85591abcc4a1d758b2c351f714d58Brazilian biomedical science faces reproducibility testde Oliveira Andrade, RodrigoNature (London, United Kingdom) (2019), 569 (7756), 318-319CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Researchers at more than 60 Brazilian labs will assess the replicability of research by their country's scientists.
- 10Sadowski, M. I., Grant, C., and Fell, T. S. (2016) Harnessing QbD, Programming Languages, and Automation for Reproducible Biology. Trends Biotechnol. 34 (3), 214– 227, DOI: 10.1016/j.tibtech.2015.11.006Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFOqtrzN&md5=d88c95c8e58a670ae8df2d051177f0a4Harnessing QbD, Programming Languages, and Automation for Reproducible BiologySadowski, Michael I.; Grant, Chris; Fell, Tim S.Trends in Biotechnology (2016), 34 (3), 214-227CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)Building robust manufg. processes from biol. components is a task that is highly complex and requires sophisticated tools to describe processes, inputs, and measurements and administrate management of knowledge, data, and materials. We argue that for bioengineering to fully access biol. potential, it will require application of statistically designed expts. to derive detailed empirical models of underlying systems. This requires execution of large-scale structured experimentation for which lab. automation is necessary. This requires development of expressive, high-level languages that allow reusability of protocols, characterization of their reliability, and a change in focus from implementation details to functional properties. We review recent developments in these areas and identify what we believe is an exciting trend that promises to revolutionize biotechnol.
- 11Shipman, S. L., Nivala, J., Macklis, J. D., and Church, G. M. (2017) CRISPR-Cas Encoding of a Digital Movie into the Genomes of a Population of Living Bacteria. Nature (London, U. K.) 547, 345– 349, DOI: 10.1038/nature23017Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFOjtrfN&md5=cb454080d420da8a7b485dc1cb54a0f1CRISPR-Cas encoding of a digital movie into the genomes of a population of living bacteriaShipman, Seth L.; Nivala, Jeff; Macklis, Jeffrey D.; Church, George M.Nature (London, United Kingdom) (2017), 547 (7663), 345-349CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)DNA is an excellent medium for archiving data. Recent efforts have illustrated the potential for information storage in DNA using synthesized oligonucleotides assembled in vitro. A relatively unexplored avenue of information storage in DNA is the ability to write information into the genome of a living cell by the addn. of nucleotides over time. Using the Cas1-Cas2 integrase, the CRISPR-Cas microbial immune system stores the nucleotide content of invading viruses to confer adaptive immunity. When harnessed, this system has the potential to write arbitrary information into the genome. Here we use the CRISPR-Cas system to encode the pixel values of black and white images and a short movie into the genomes of a population of living bacteria. In doing so, we push the tech. limits of this information storage system and optimize strategies to minimize those limitations. We also uncover underlying principles of the CRISPR-Cas adaptation system, including sequence determinants of spacer acquisition that are relevant for understanding both the basic biol. of bacterial adaptation and its technol. applications. This work demonstrates that this system can capture and stably store practical amts. of real data within the genomes of populations of living cells.
- 12Mazurkiewicz, P., Tang, C. M., Boone, C., and Holden, D. W. (2006) Signature-Tagged Mutagenesis: Barcoding Mutants for Genome-Wide Screens. Nat. Rev. Genet. 7, 929– 939, DOI: 10.1038/nrg1984Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1KisrzI&md5=531e666853dc851503771f9e5a0af78cSignature-tagged mutagenesis: barcoding mutants for genome-wide screensMazurkiewicz, Piotr; Tang, Christoph M.; Boone, Charles; Holden, David W.Nature Reviews Genetics (2006), 7 (12), 929-939CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A review. DNA signature tags (mol. barcodes) facilitate functional screens by identifying mutants in mixed populations that have a reduced or increased adaptation to a particular environment. Many innovative adaptations and refinements in the technol. have been described since its original use with Salmonella; they have yielded a wealth of information on a broad range of biol. processes - mainly in bacteria, but also in yeast and other fungi, viruses, parasites and, most recently, in mammalian cells. By combining whole-genome microarrays and comprehensive ordered libraries of mutants, high-throughput functional screens can now be achieved on a genomic scale.
- 13Liu, H., Price, M. N., Waters, R. J., Ray, J., Carlson, H. K., Lamson, J. S., Chakraborty, R., Arkin, A. P., and Deutschbauer, A. M. (2018) Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria. mSystems 3, e00143– 17, DOI: 10.1128/mSystems.00143-17Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVentbjJ&md5=026479c912cf192d19d0ad6e7c2a7a10Magic pools: parallel assessment of transposon delivery vectors in bacteriaLiu, Hualan; Price, Morgan N.; Waters, Robert Jordan; Ray, Jayashree; Carlson, Hans K.; Lamson, Jacob S.; Chakraborty, Romy; Arkin, Adam P.; Deutschbauer, Adam M.mSystems (2018), 3 (1), e00143-17/1-e00143-17/17CODEN: MSYSBR; ISSN:2379-5077. (American Society for Microbiology)Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term "magic pools." Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis expts. To identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylum Bacteroidetes. IMPORTANCE Mol. genetics is indispensable for interrogating the physiol. of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a "magic pool." The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA "parts," we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.
- 14Yu, C., Mannan, A. M., Metta Yvone, G., Ross, K. N., Zhang, Y.-L., Marton, M. A., Taylor, B. R., Crenshaw, A., Gould, J. Z., and Tamayo, P. (2016) High-Throughput Identification of Genotype-Specific Cancer Vulnerabilities in Mixtures of Barcoded Tumor Cell Lines. Nat. Biotechnol. 34, 419, DOI: 10.1038/nbt.3460Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XivFKgsro%253D&md5=8c5206e6e3dd2fe19673acc9eed04956High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell linesYu, Channing; Mannan, Aristotle M.; Yvone, Griselda Metta; Ross, Kenneth N.; Zhang, Yan-Ling; Marton, Melissa A.; Taylor, Bradley R.; Crenshaw, Andrew; Gould, Joshua Z.; Tamayo, Pablo; Weir, Barbara A.; Tsherniak, Aviad; Wong, Bang; Garraway, Levi A.; Shamji, Alykhan F.; Palmer, Michelle A.; Foley, Michael A.; Winckler, Wendy; Schreiber, Stuart L.; Kung, Andrew L.; Golub, Todd R.Nature Biotechnology (2016), 34 (4), 419-423CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large nos. of compds. against large nos. of cell lines is currently impractical, and such expts. are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixts. of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compds., PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo.
- 15Bhang, H.-E. C., Ruddy, D. A., Krishnamurthy Radhakrishna, V., Caushi, J. X., Zhao, R., Hims, M. M., Singh, A. P., Kao, I., Rakiec, D., Shaw, P. (2015) Studying Clonal Dynamics in Response to Cancer Therapy Using High-Complexity Barcoding. Nat. Med. 21 (5), 440– 448, DOI: 10.1038/nm.3841Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXnvFSqu7o%253D&md5=4d5c7c32cb8a717bdb77e86c6919a554Studying clonal dynamics in response to cancer therapy using high-complexity barcodingBhang, Hyo-eun C.; Ruddy, David A.; Krishnamurthy Radhakrishna, Viveksagar; Caushi, Justina X.; Zhao, Rui; Hims, Matthew M.; Singh, Angad P.; Kao, Iris; Rakiec, Daniel; Shaw, Pamela; Balak, Marissa; Raza, Alina; Ackley, Elizabeth; Keen, Nicholas; Schlabach, Michael R.; Palmer, Michael; Leary, Rebecca J.; Chiang, Derek Y.; Sellers, William R.; Michor, Franziska; Cooke, Vesselina G.; Korn, Joshua M.; Stegmeier, FrankNature Medicine (New York, NY, United States) (2015), 21 (5), 440-448CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)Resistance to cancer therapies presents a significant clin. challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resoln. limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resoln. tracking of more than 1 million cancer cells under drug treatment. In two clin. relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quant. assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.
- 16McKenna, A., Findlay, G. M., Gagnon, J. A., Horwitz, M. S., Schier, A. F., and Shendure, J. (2016) Whole-Organism Lineage Tracing by Combinatorial and Cumulative Genome Editing. Science (Washington, DC, U. S.) 353 (6298), aaf7907, DOI: 10.1126/science.aaf7907Google ScholarThere is no corresponding record for this reference.
- 17Plesa, C., Sidore, A. M., Lubock, N. B., Zhang, D., and Kosuri, S. (2018) Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes. Science (Washington, DC, U. S.) 359, 343– 347, DOI: 10.1126/science.aao5167Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFKntrs%253D&md5=2618042dc2d0175754d9555a2a4176f7Multiplexed gene synthesis in emulsions for exploring protein functional landscapesPlesa, Calin; Sidore, Angus M.; Lubock, Nathan B.; Zhang, Di; Kosuri, SriramScience (Washington, DC, United States) (2018), 359 (6373), 343-347CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Improving our ability to construct and functionally characterize DNA sequences would broadly accelerate progress in biol. Here, we introduce DropSynth, a scalable, low-cost method to build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene's assembly, which are then processed and assembled in water-in-oil emulsions. We used DropSynth to successfully build more than 7000 synthetic genes that encode phylogenetically diverse homologs of two essential genes in Escherichia coli. We tested the ability of phosphopantetheine adenylyltransferase homologs to complement a knockout E. coli strain in multiplex, revealing core functional motifs and reasons underlying homolog incompatibility. DropSynth coupled with multiplexed functional assays allows us to rationally explore sequence-function relationships at an unprecedented scale.
- 18Woodruff, L. B. A., Gorochowski, T. E., Roehner, N., Mikkelsen, T. S., Densmore, D., Gordon, D. B., Nicol, R., and Voigt, C. A. (2016) Registry in a Tube: Multiplexed Pools of Retrievable Parts for Genetic Design Space Exploration. Nucleic Acids Res. 45 (3), 1553– 1565, DOI: 10.1093/nar/gkw1226Google ScholarThere is no corresponding record for this reference.
- 19Zimmermann, G. and Neri, D. (2016) DNA-Encoded Chemical Libraries: Foundations and Applications in Lead Discovery. Drug Discovery Today 21 (11), 1828– 1834, DOI: 10.1016/j.drudis.2016.07.013Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1ymsbvK&md5=112af87dee3a38ebf41d1a094943cbc7DNA-encoded chemical libraries: foundations and applications in lead discoveryZimmermann, Gunther; Neri, DarioDrug Discovery Today (2016), 21 (11), 1828-1834CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)A review. DNA-encoded chem. libraries have emerged as a powerful tool for hit identification in the pharmaceutical industry and in academia. Similar to biol. display techniques (such as phage display technol.), DNA-encoded chem. libraries contain a link between the displayed chem. building block and an amplifiable genetic barcode on DNA. Using routine procedures, libraries contg. millions to billions of compds. can be easily produced within a few weeks. The resulting compd. libraries are screened in a single test tube against proteins of pharmaceutical interest and hits can be identified by PCR amplification of DNA barcodes and subsequent high-throughput sequencing.
- 20Hawkins, J. A., Jones, S. K., Finkelstein, I. J., and Press, W. H. (2018) Indel-Correcting DNA Barcodes for High-Throughput Sequencing. Proc. Natl. Acad. Sci. U. S. A. 115 (27), E6217– E6226, DOI: 10.1073/pnas.1802640115Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFKhsbzO&md5=5f94c7f456f1c9a51fa65ee9949e184cIndel-correcting DNA barcodes for high-throughput sequencingHawkins, John A.; Jones, Stephen K., Jr.; Finkelstein, Ilya J.; Press, William H.Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (27), E6217-E6226CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Many large-scale, high-throughput expts. use DNA barcodes, short DNA sequences prepended to DNA libraries, for identification of individuals in pooled biomol. populations. However, DNA synthesis and sequencing errors confound the correct interpretation of obsd. barcodes and can lead to significant data loss or spurious results. Widely used error-correcting codes borrowed from computer science (e.g., Hamming, Levenshtein codes) do not properly account for insertions and deletions (indels) in DNA barcodes, even though deletions are the most common type of synthesis error. Here, we present and exptl. validate filled/truncated right end edit (FREE) barcodes, which correct substitution, insertion, and deletion errors, even when these errors alter the barcode length. FREE barcodes are designed with exptl. considerations in mind, including balanced guanine-cytosine (GC) content, minimal homopolymer runs, and reduced internal hairpin propensity. We generate and include lists of barcodes with different lengths and error correction levels that may be useful in diverse high-throughput applications, including >106 single-error-correcting 16-mers that strike a balance between decoding accuracy, barcode length, and library size. Moreover, concatenating two or more FREE codes into a single barcode increases the available barcode space combinatorially, generating lists with >1015 error correcting barcodes. The included software for creating barcode libraries and decoding sequenced barcodes is efficient and designed to be user-friendly for the general biol. community.
- 21de Lorenzo, V. and Schmidt, M. (2018) Biological Standards for the Knowledge-Based BioEconomy: What Is at Stake. New Biotechnol. 40, 170– 180, DOI: 10.1016/j.nbt.2017.05.001Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXns1yiu7k%253D&md5=1a1ed189419432ce650a512bf347b1aeBiological standards for the Knowledge-Based BioEconomy: What is at stakede Lorenzo, Victor; Schmidt, MarkusNew Biotechnology (2018), 40 (Part_A), 170-180CODEN: NBEIBR; ISSN:1871-6784. (Elsevier B.V.)The contribution of life sciences to the Knowledge-Based Bioeconomy (KBBE) asks for the transition of contemporary, gene-based biotechnol. from being a trial-and-error endeavour to becoming an authentic branch of engineering. One requisite to this end is the need for stds. to measure and represent accurately biol. functions, along with languages for data description and exchange. However, the inherent complexity of biol. systems and the lack of quant. tradition in the field have largely curbed this enterprise. Fortunately, the onset of systems and synthetic biol. has emphasized the need for stds. not only to manage omics data, but also to increase reproducibility and provide the means of engineering living systems in earnest. Some domains of biotechnol. can be easily standardized (e.g. phys. compn. of DNA sequences, tools for genome editing, languages to encode workflows), while others might be standardized with some dedicated research (e.g. biol. metrol., operative systems for bio-programming cells) and finally others will require a considerable effort, e.g. defining the rules that allow functional compn. of biol. activities. Despite difficulties, these are worthy attempts, as the history of technol. shows that those who set/adopt stds. gain a competitive advantage over those who do not.
- 22Schmidt, M. and de Lorenzo, V. (2012) Synthetic Constructs in/for the Environment: Managing the Interplay between Natural and Engineered Biology. FEBS Lett. 586 (15), 2199– 2206, DOI: 10.1016/j.febslet.2012.02.022Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjsVWnsLg%253D&md5=b87879583ccb78993d7c707475ea2769Synthetic constructs in/for the environment: Managing the interplay between natural and engineered biologySchmidt, Markus; de Lorenzo, VictorFEBS Letters (2012), 586 (15), 2199-2206CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. The plausible release of deeply engineered or even entirely synthetic/artificial microorganisms raises the issue of their intentional (e.g., bioremediation) or accidental interaction with the environment. Containment systems designed in the 1980s-1990s for limiting the spread of genetically engineered bacteria and their recombinant traits are still applicable to contemporary synthetic biol. constructs. Yet, the ease of DNA synthesis and the uncertainty on how non-natural properties and strains could interplay with the existing biol. world poses yet again the challenge of designing safe and efficacious firewalls to curtail possible interactions. Such barriers may include xeno-nucleic acids (XNAs) instead of DNA as information-bearing mols., rewriting the genetic code to make it non-understandable by the existing gene expression machineries, and/or making growth dependent on xenobiotic chems.
- 23Lin, Z., Deng, B., Jiao, Z., Wu, B., Xu, X., Yu, D., and Li, W. (2013) A Versatile Mini-MazF-Cassette for Marker-Free Targeted Genetic Modification in Bacillus Subtilis. J. Microbiol. Methods 95, 207– 214, DOI: 10.1016/j.mimet.2013.07.020Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsleltrrK&md5=36e4e22d5cf48af7a009dcb35da7f9e9A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilisLin, Zhiwei; Deng, Bin; Jiao, Zhihua; Wu, Bingbing; Xu, Xin; Yu, Dongyou; Li, WeifenJournal of Microbiological Methods (2013), 95 (2), 207-214CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier B.V.)There are some drawbacks for the MazF-cassette constructed in previous reports for marker-free genetic manipulation in Bacillus subtilis, including cloning-dependent methodol. and a non-strictly controlled expression system. In this study, modifications on the mazF-cassette are carried out, such as using mini zeocin resistance gene as pos.-selectable marker and strictly controlled xyl promoter from the B. subtilis to replace non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium. Then the mini-mazF-cassette was successfully applied to knock-out the amyE gene, to delete a 90-kb gene cluster, and to knock-in a green fluorescent protein expression cassette employing a cloning-independent methodol., without introducing undesirable redundant sequences at the modified locus in the B. subtilis 1A751. Also, the mini-mazF-cassette could be used repeatedly to delete multiple genes or gene clusters with only a 2- to 2.5-kb PCR-fused fragment, which largely reduced the frequency of nucleic acid mutations generated by PCR compared to previous reports. We further demonstrated that the frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%. The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3 days. This modified cassette has remarkable improvement compared to existing approaches and is applicable for available manipulating Bacillus species chromosomes.
- 24Datsenko, K. A., Wanner, B. L., and Beckwith, J. (2000) One-Step Inactivation of Chromosomal Genes in Escherichia Coli K-12 Using PCR Products. Proc. Natl. Acad. Sci. U. S. A. 97 (12), 6640– 6645, DOI: 10.1073/pnas.120163297Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXktFais7c%253D&md5=85388def19d7c14ddada4b70a0bec1eeOne-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR productsDatsenko, Kirill A.; Wanner, Barry L.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (12), 6640-6645CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The authors have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homol. to the targeted gene(s). In this procedure, recombination requires the phage λ Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy no. plasmid. To demonstrate the utility of this approach, the authors generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the resp. PCR products, the authors made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, esp. in genome anal. of E. coli and other bacteria because the procedure can be done in wild-type cells.
- 25Li, Y., Lin, Z., Huang, C., Zhang, Y., Wang, Z., Tang, Y., Chen, T., and Zhao, X. (2015) Metabolic Engineering of Escherichia Coli Using CRISPR–Cas9Meditated Genome Editing. Metab. Eng. 31, 13– 21, DOI: 10.1016/j.ymben.2015.06.006Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFaiurjM&md5=1285f024ef6155504519f691e0d83edfMetabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editingLi, Yifan; Lin, Zhenquan; Huang, Can; Zhang, Yan; Wang, Zhiwen; Tang, Ya-jie; Chen, Tao; Zhao, XuemingMetabolic Engineering (2015), 31 (), 13-21CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)Engineering cellular metab. for improved prodn. of valuable chems. requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli. This system enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously. We also found that cells with intact mismatch repair system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential, we used our method to integrate the β-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate (MEP) pathway and central metabolic pathways for β-carotene overprodn. We collectively tested 33 genomic modifications and constructed more than 100 genetic variants for combinatorially exploring the metabolic landscape. Our best producer contained 15 targeted mutations and produced 2.0 g/L β-carotene in fed-batch fermn.
- 26Koo, B. M., Kritikos, G., Farelli, J. D., Todor, H., Tong, K., Kimsey, H., Wapinski, I., Galardini, M., Cabal, A., Peters, J. M. (2017) Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus Subtilis. Cell Syst. 22, 291– 305, DOI: 10.1016/j.cels.2016.12.013Google ScholarThere is no corresponding record for this reference.
- 27Altenbuchner, J. (2016) Editing of the Bacillus Subtilis Genome by the CRISPR-Cas9 System. Appl. Environ. Microbiol. 82 (17), 5421– 5427, DOI: 10.1128/AEM.01453-16Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVahurvJ&md5=ede5e7d6c146a01bbac9c39f4872a929Editing of the Bacillus subtilis genome by the CRISPR-Cas9 systemAltenbuchner, JosefApplied and Environmental Microbiology (2016), 82 (17), 5421-5427CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)The clustered regularly interspaced short palindromic repeat (CRISPR)-assocd. (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temp.-sensitive replication origin of plasmid pE194ts for B.subtilis, and a kanamycin resistance gene working in both organisms. This sgRNA guides the Cas9 nuclease to its target. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Repair of the DSBs and the required modification of the genome are achieved by adding homol. templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homol. templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B.subtilis chromosome and by repair of the trpC2 mutation of B.subtilis 168. For genome editing, it carries the cas9 gene under the control of the B.subtilis mannose-inducible promoter PmanP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. The 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site.
- 28Veening, J.-W., Murray, H., and Errington, J. (2009) A Mechanism for Cell Cycle Regulation of Sporulation Initiation in Bacillus Subtilis. Genes Dev. 23, 1959– 1970, DOI: 10.1101/gad.528209Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVegurbK&md5=0b50b569cd9816014dcfd87e2d730258A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilisVeening, Jan-Willem; Murray, Heath; Errington, JeffGenes & Development (2009), 23 (16), 1959-1970CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)Coordination of DNA replication with cellular development is a crucial problem in most living organisms. Bacillus subtilis cells switch from vegetative growth to sporulation when starved. Sporulation normally occurs in cells that have stopped replicating DNA and have two completed chromosomes, one destined for the prespore and the other for the mother cell. It has long been recognized that there is a sensitive period in the cell cycle during which the initiation of spore development can be triggered, presumably to allow for the generation of exactly two complete chromosomes. However, the mechanism responsible for this has remained unclear. Here we show that the sda gene, previously identified as a checkpoint factor preventing sporulation in response to DNA damage, exerts cell cycle control over the initiation of sporulation. Expression of sda occurs in a pulsatile manner, with a burst of expression each cell cycle at the onset of DNA replication. Up-regulation of the intrinsically unstable Sda protein, which is dependent on the active form of the DNA replication initiator protein, DnaA, transiently inhibits the initiation of sporulation. This regulation avoids the generation of spore formers with replicating chromosomes, which would result in diploid or polyploid spores that we show have reduced viability.
- 29Kobayashi, K., Ehrlich, S. D., Albertini, A., Amati, G., Andersen, K. K., Arnaud, M., Asai, K., Ashikaga, S., Aymerich, S., Bessieres, P. (2003) Essential Bacillus Subtilis Genes. Proc. Natl. Acad. Sci. U. S. A. 100 (8), 4678– 4683, DOI: 10.1073/pnas.0730515100Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjt12ns7o%253D&md5=8c03de4a0dd3ddf96ae5575d372346b6Essential bacillus subtilis genesKobayashi, K.; Ehrlich, S. D.; Albertini, A.; Amati, G.; Andersen, K. K.; Arnaud, M.; Asai, K.; Ashikaga, S.; Aymerich, S.; Bessieres, P.; Boland, F.; Brignell, S. C.; Bron, S.; Bunai, K.; Chapuis, J.; Christiansen, L. C.; Danchin, A.; Debarbouille, M.; Dervyn, E.; Deuerling, E.; Devine, K.; Devine, S. K.; Dreesen, O.; Errington, J.; Fillinger, S.; Foster, S. J.; Fujita, Y.; Galizzi, A.; Gardan, R.; Eschevins, C.; Fukushima, T.; Haga, K.; Harwood, C. R.; Hecker, M.; Hosoya, D.; Hullo, M. F.; Kakeshita, H.; Karamata, D.; Kasahara, Y.; Kawamura, F.; Koga, K.; Koski, P.; Kuwana, R.; Imamura, D.; Ishimaru, M.; Ishikawa, S.; Ishio, I.; Le Coq, D.; Masson, A.; Mauel, C.; Meima, R.; Mellado, R. P.; Moir, A.; Moriya, S.; Nagakawa, E.; Nanamiya, H.; Nakai, S.; Nygaard, P.; Ogura, M.; Ohanan, T.; O'Reilly, M.; O'Rourke, M.; Pragai, Z.; Pooley, H. M.; Rapoport, G.; Rawlins, J. P.; Rivas, L. A.; Rivolta, C.; Sadaie, A.; Sadaie, Y.; Sarvas, M.; Sato, T.; Saxild, H. H.; Scanlan, E.; Schumann, W.; Seegers, J. F. M. L.; Sekiguchi, J.; Sekowska, A.; Seror, S. J.; Simon, M.; Stragier, P.; Studer, R.; Takamatsu, H.; Tanaka, T.; Takeuchi, M.; Thomaides, H. B.; Vagner, V.; van Dijl, J. M.; Watabe, K.; Wipat, A.; Yamamoto, H.; Yamamoto, M.; Yamamoto, Y.; Yamane, K.; Yata, K.; Yoshida, K.; Yoshikawa, H.; Zuber, U.; Ogasawara, N.Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (8), 4678-4683CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)To est. the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ≈4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metab., with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the detn. of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.
- 30Juhas, M., Reuß, D. R., Zhu, B., and Commichau, F. M. (2014) Bacillus Subtilis and Escherichia Coli Essential Genes and Minimal Cell Factories after One Decade of Genome Engineering. Microbiology (London, U. K.) 160, 2341– 2351, DOI: 10.1099/mic.0.079376-0Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXktVyjuw%253D%253D&md5=041b4443f4ab77c644edeed44e58036eBacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineeringJuhas, Mario; Reuss, Daniel R.; Zhu, Bingyao; Commichau, Fabian M.Microbiology (Reading, United Kingdom) (2014), 160 (11), 2341-2351CODEN: MROBEO; ISSN:1350-0872. (Society for General Microbiology)A review. Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell "chassis". Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnol. applications. Detn. of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome redn. analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.
- 31Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., and Yang, S. (2015) Multigene Editing in the Escherichia Coli Genome via the CRISPR-Cas9 System. Appl. Environ. Microbiol. 81 (7), 2506– 2514, DOI: 10.1128/AEM.04023-14Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvV2hsbY%253D&md5=7738bd7921f45b04f0f3dcac4d99a4d8Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 systemJiang, Yu; Chen, Biao; Duan, Chunlan; Sun, Bingbing; Yang, Junjie; Yang, ShengApplied and Environmental Microbiology (2015), 81 (7), 2506-2514CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.
- 32Sung, W., Ackerman, M. S., Gout, J.-F., Miller, S. F., Williams, E., Foster, P. L., and Lynch, M. (2015) Asymmetric Context-Dependent Mutation Patterns Revealed through Mutation-Accumulation Experiments. Mol. Biol. Evol. 32 (7), 1672– 1683, DOI: 10.1093/molbev/msv055Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs12rsL%252FP&md5=56462d5a5628bf8c6550e9b490aa0b25Asymmetric context-dependent mutation patterns revealed through mutation-accumulation experimentsSung, Way; Ackerman, Matthew S.; Gout, Jean-Francois; Miller, Samuel F.; Williams, Emily; Foster, Patricia L.; Lynch, MichaelMolecular Biology and Evolution (2015), 32 (7), 1672-1683CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)Despite the general assumption that site-specific mutation rates are independent of the local sequence context, a growing body of evidence suggests otherwise. To further examine context-dependent patterns of mutation, we amassed 5,645 spontaneous mutations in wild- type (WT) and mismatch-repair deficient (MMR-) mutation-accumulation (MA) lines of the gram-pos. model organism Bacillus subtilis. We then analyzed >7,500 spontaneous base-substitution mutations across B. subtilis, Escherichia coli, and Mesoplasma florum WT and MMR- MA lines, finding a context-dependent mutation pattern that is asym. around the origin of replication. Different neighboring nucleotides can alter site-specific mutation rates by as much as 75-fold, with sites neighboring G:C base pairs or dimers involving alternating pyrimidine-purine and purine-pyrimidine nucleotides having significantly elevated mutation rates. The influence of context-dependent mutation on genome architecture is strongest in M. florum, consistent with the reduced efficiency of selection in organisms with low effective population size. If not properly accounted for, the disparities arising from patterns of context-dependent mutation can significantly influence interpretations of pos. and purifying selection.
- 33Lee, H., Popodi, E., Tang, H., and Foster, P. L. (2012) Rate and Molecular Spectrum of Spontaneous Mutations in the Bacterium Escherichia Coli as Determined by Whole-Genome Sequencing. Proc. Natl. Acad. Sci. U. S. A. 109 (41), E2774– E2783, DOI: 10.1073/pnas.1210309109Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFKntbjM&md5=e44fb70553c7eadc11516ee3346dcd4dRate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencingLee, Heewook; Popodi, Ellen; Tang, Haixu; Foster, Patricia L.Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (41), E2774-E2783, SE2774/1-SE2774/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and mol. processes. In this report, we analyze spontaneous mutations accumulated over thousands of generations by wild-type Escherichia coli and a deriv. defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. col strain is ∼1 × 10-3 per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5'ApC3'/3'TpG5' sites (where bases 5'A and 3'T are mutated) and, to a lesser extent, at 5'GpC3'/3'CpG5' sites (where bases 5'G and 3'C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that det. mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions.
- 34Wang, K., de la Torre, D., Robertson, W. E., and Chin, J. W. (2019) Programmed Chromosome Fission and Fusion Enable Precise Large-Scale Genome Rearrangement and Assembly. Science (Washington, DC, U. S.) 365, 922– 926, DOI: 10.1126/science.aay0737Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslSqsbvO&md5=aa1e8a441a5d31f7da1c52fc51864646Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assemblyWang, Kaihang; de la Torre, Daniel; Robertson, Wesley E.; Chin, Jason W.Science (Washington, DC, United States) (2019), 365 (6456), 922-926CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The model bacterium Escherichia coli has a single circular chromosome. Wang et al. created a method to fragment the E. coli genome into independent chromosomes that can be modified, rearranged, and recombined. The efficient fission of the unmodified E. coli genome into two defined, stable pairs of synthetic chromosomes provides common intermediates for large-scale genome manipulations such as inversion and translocation. Fusion of synthetic chromosomes from distinct cells generated a single genome in a target cell. Precise, rapid, large-scale genome engineering operations are useful tools for creating diverse synthetic genomes.
- 35Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A. (2019) Search-and-Replace Genome Editing without Double-Strand Breaks or Donor DNA. Nature (London, U. K.) 576, 149– 157, DOI: 10.1038/s41586-019-1711-4Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFGns7rO&md5=1fbd690d78a81c3b90ad5f9dad5be086Search-and-replace genome editing without double-strand breaks or donor DNAAnzalone, Andrew V.; Randolph, Peyton B.; Davis, Jessie R.; Sousa, Alexander A.; Koblan, Luke W.; Levy, Jonathan M.; Chen, Peter J.; Wilson, Christopher; Newby, Gregory A.; Raguram, Aditya; Liu, David R.Nature (London, United Kingdom) (2019), 576 (7785), 149-157CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homol.-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants assocd. with human diseases.
- 36Velázquez, E., Lorenzo, V. de, and Al-Ramahi, Y. (2019) Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery. ACS Synth. Biol. 8 (9), 2186– 2193, DOI: 10.1021/acssynbio.9b00293Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsF2ntbzN&md5=f06edd98e918993b500d50caa1978a9cRecombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron DeliveryVelazquez, Elena; Lorenzo, Victor de; Al-Ramahi, YamalACS Synthetic Biology (2019), 8 (9), 2186-2193CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Group II introns were developed time ago as tools for the construction of knockout mutants in a wide range of organisms, ranging from Gram-pos. and Gram-neg. bacteria to human cells. Utilizing these introns is advantageous because they are independent of the host's DNA recombination machinery, they can carry heterologous sequences (and thus be used as vehicles for gene delivery), and they can be easily retargeted for subsequent insertions of addnl. genes at the user's will. Alas, the use of this platform has been limited, as insertion efficiencies greatly change depending on the target sites and cannot be predicted a priori. Moreover, the ability of introns to perform their own splicing and integration is compromised when they carry foreign sequences. To overcome these limitations, we merged the group II intron-based TargeTron system with CRISPR/Cas9 counter-selection. To this end, we first engineered a new group-II intron by replacing the retrotransposition-activated selectable marker (RAM) with ura3 and retargeting it to a new site in the lacZ gene of E. coli. Then, we showed proved that directing CRISPR/Cas9 towards the wild-type sequences dramatically increased the chances of finding clones that integrated the retrointron into the target lacZ sequence. The CRISPR-Cas9 counter selection strategy presented herein thus overcomes a major limitation that has prevented the use of group II introns as devices for gene delivery and genome editing at large in a recombination-independent fashion.
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Abstract
Figure 1
Figure 1. An example of version control in strain engineering. A repository with the master culture (red) is branched by two different biologists to create new Strain 1 (purple) and Strain 3 (green). Each dot in the commit tree represents a strain engineering milestone and is created by modifying the parent strand (or an earlier derivative). For critical (user defined) steps in the engineering process, a unique identifier can be generated and stored as a DNA barcode in the strain chromosome. The barcode is associated with a “commit” within our version control system, with each commit containing the digital footprint (files and other metadata information) that lead to this milestone.
Figure 2
Figure 2. Writing a barcode (Top): During strain engineering, a key milestone is reached, and the strain is barcoded with a unique DNA sequence generated by the CellRepo website. A commit to the repository is made connecting the unique barcode (now in the chromosome of the cell) with all the cell’s related documentation. Reading a barcode (Center): A DNA barcode is read from a sample strain enabling the lookup of all the strains’ documentation from the CellRepo repository. Updating a barcode (Bottom): Strain 1 was barcoded with Barcode-1. When the strain is modified to give Strain 2 a new barcode must be used. The strain needs to be updated with Barcode-2 replacing Barcode-1. The repository keeps track of the barcode’s “lineage”; thus, via sequencing Barcode-2 one can trace back the parental strain (and its own barcode).
Figure 3
Figure 3. Graphic representation of the barcoding sites for both species. Orange lines show homologous regions used for barcoding purposes. Coordinates indicate start and end of nearby genes. Green blocks correspond to universal primer binding sites (25 bp). Yellow and red blocks represent synchronization (9 bp) and checksum sequences (18 bp) respectively (Supporting Information). For E. coli and B. subtilis, NCBI genome Accession number CP009273.1 and AL009126.3 were used, respectively.
Figure 4
Figure 4. Screenshot of the repository summary page. The list of changes shows all steps made in the process of engineering a Bacillus subtilis mutant strain with inserted GFP gene and represents a digital footprint of the cell line. Two key engineering milestones (revision 4 and 10) have been linked via a genetic barcode to the cell line.
Figure 5
Figure 5. Examples of information stored in the repository: (a) essential strain details, (b) list of added files that are part of the digital documentation for a cell line at a given point in time, and (c) list of revisions barcoded into the cell line, called “snapshots”, as they uniquely link to the digital documentation (i.e., the digital twin) at a specific point in the cell engineering cycle. In this example revision with id r10:acc4afc32349 and r4:070c713ad448 have been barcoded back into the cell line.
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- 18Woodruff, L. B. A., Gorochowski, T. E., Roehner, N., Mikkelsen, T. S., Densmore, D., Gordon, D. B., Nicol, R., and Voigt, C. A. (2016) Registry in a Tube: Multiplexed Pools of Retrievable Parts for Genetic Design Space Exploration. Nucleic Acids Res. 45 (3), 1553– 1565, DOI: 10.1093/nar/gkw1226There is no corresponding record for this reference.
- 19Zimmermann, G. and Neri, D. (2016) DNA-Encoded Chemical Libraries: Foundations and Applications in Lead Discovery. Drug Discovery Today 21 (11), 1828– 1834, DOI: 10.1016/j.drudis.2016.07.01319https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1ymsbvK&md5=112af87dee3a38ebf41d1a094943cbc7DNA-encoded chemical libraries: foundations and applications in lead discoveryZimmermann, Gunther; Neri, DarioDrug Discovery Today (2016), 21 (11), 1828-1834CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Ltd.)A review. DNA-encoded chem. libraries have emerged as a powerful tool for hit identification in the pharmaceutical industry and in academia. Similar to biol. display techniques (such as phage display technol.), DNA-encoded chem. libraries contain a link between the displayed chem. building block and an amplifiable genetic barcode on DNA. Using routine procedures, libraries contg. millions to billions of compds. can be easily produced within a few weeks. The resulting compd. libraries are screened in a single test tube against proteins of pharmaceutical interest and hits can be identified by PCR amplification of DNA barcodes and subsequent high-throughput sequencing.
- 20Hawkins, J. A., Jones, S. K., Finkelstein, I. J., and Press, W. H. (2018) Indel-Correcting DNA Barcodes for High-Throughput Sequencing. Proc. Natl. Acad. Sci. U. S. A. 115 (27), E6217– E6226, DOI: 10.1073/pnas.180264011520https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFKhsbzO&md5=5f94c7f456f1c9a51fa65ee9949e184cIndel-correcting DNA barcodes for high-throughput sequencingHawkins, John A.; Jones, Stephen K., Jr.; Finkelstein, Ilya J.; Press, William H.Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (27), E6217-E6226CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Many large-scale, high-throughput expts. use DNA barcodes, short DNA sequences prepended to DNA libraries, for identification of individuals in pooled biomol. populations. However, DNA synthesis and sequencing errors confound the correct interpretation of obsd. barcodes and can lead to significant data loss or spurious results. Widely used error-correcting codes borrowed from computer science (e.g., Hamming, Levenshtein codes) do not properly account for insertions and deletions (indels) in DNA barcodes, even though deletions are the most common type of synthesis error. Here, we present and exptl. validate filled/truncated right end edit (FREE) barcodes, which correct substitution, insertion, and deletion errors, even when these errors alter the barcode length. FREE barcodes are designed with exptl. considerations in mind, including balanced guanine-cytosine (GC) content, minimal homopolymer runs, and reduced internal hairpin propensity. We generate and include lists of barcodes with different lengths and error correction levels that may be useful in diverse high-throughput applications, including >106 single-error-correcting 16-mers that strike a balance between decoding accuracy, barcode length, and library size. Moreover, concatenating two or more FREE codes into a single barcode increases the available barcode space combinatorially, generating lists with >1015 error correcting barcodes. The included software for creating barcode libraries and decoding sequenced barcodes is efficient and designed to be user-friendly for the general biol. community.
- 21de Lorenzo, V. and Schmidt, M. (2018) Biological Standards for the Knowledge-Based BioEconomy: What Is at Stake. New Biotechnol. 40, 170– 180, DOI: 10.1016/j.nbt.2017.05.00121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXns1yiu7k%253D&md5=1a1ed189419432ce650a512bf347b1aeBiological standards for the Knowledge-Based BioEconomy: What is at stakede Lorenzo, Victor; Schmidt, MarkusNew Biotechnology (2018), 40 (Part_A), 170-180CODEN: NBEIBR; ISSN:1871-6784. (Elsevier B.V.)The contribution of life sciences to the Knowledge-Based Bioeconomy (KBBE) asks for the transition of contemporary, gene-based biotechnol. from being a trial-and-error endeavour to becoming an authentic branch of engineering. One requisite to this end is the need for stds. to measure and represent accurately biol. functions, along with languages for data description and exchange. However, the inherent complexity of biol. systems and the lack of quant. tradition in the field have largely curbed this enterprise. Fortunately, the onset of systems and synthetic biol. has emphasized the need for stds. not only to manage omics data, but also to increase reproducibility and provide the means of engineering living systems in earnest. Some domains of biotechnol. can be easily standardized (e.g. phys. compn. of DNA sequences, tools for genome editing, languages to encode workflows), while others might be standardized with some dedicated research (e.g. biol. metrol., operative systems for bio-programming cells) and finally others will require a considerable effort, e.g. defining the rules that allow functional compn. of biol. activities. Despite difficulties, these are worthy attempts, as the history of technol. shows that those who set/adopt stds. gain a competitive advantage over those who do not.
- 22Schmidt, M. and de Lorenzo, V. (2012) Synthetic Constructs in/for the Environment: Managing the Interplay between Natural and Engineered Biology. FEBS Lett. 586 (15), 2199– 2206, DOI: 10.1016/j.febslet.2012.02.02222https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjsVWnsLg%253D&md5=b87879583ccb78993d7c707475ea2769Synthetic constructs in/for the environment: Managing the interplay between natural and engineered biologySchmidt, Markus; de Lorenzo, VictorFEBS Letters (2012), 586 (15), 2199-2206CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. The plausible release of deeply engineered or even entirely synthetic/artificial microorganisms raises the issue of their intentional (e.g., bioremediation) or accidental interaction with the environment. Containment systems designed in the 1980s-1990s for limiting the spread of genetically engineered bacteria and their recombinant traits are still applicable to contemporary synthetic biol. constructs. Yet, the ease of DNA synthesis and the uncertainty on how non-natural properties and strains could interplay with the existing biol. world poses yet again the challenge of designing safe and efficacious firewalls to curtail possible interactions. Such barriers may include xeno-nucleic acids (XNAs) instead of DNA as information-bearing mols., rewriting the genetic code to make it non-understandable by the existing gene expression machineries, and/or making growth dependent on xenobiotic chems.
- 23Lin, Z., Deng, B., Jiao, Z., Wu, B., Xu, X., Yu, D., and Li, W. (2013) A Versatile Mini-MazF-Cassette for Marker-Free Targeted Genetic Modification in Bacillus Subtilis. J. Microbiol. Methods 95, 207– 214, DOI: 10.1016/j.mimet.2013.07.02023https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsleltrrK&md5=36e4e22d5cf48af7a009dcb35da7f9e9A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilisLin, Zhiwei; Deng, Bin; Jiao, Zhihua; Wu, Bingbing; Xu, Xin; Yu, Dongyou; Li, WeifenJournal of Microbiological Methods (2013), 95 (2), 207-214CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier B.V.)There are some drawbacks for the MazF-cassette constructed in previous reports for marker-free genetic manipulation in Bacillus subtilis, including cloning-dependent methodol. and a non-strictly controlled expression system. In this study, modifications on the mazF-cassette are carried out, such as using mini zeocin resistance gene as pos.-selectable marker and strictly controlled xyl promoter from the B. subtilis to replace non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium. Then the mini-mazF-cassette was successfully applied to knock-out the amyE gene, to delete a 90-kb gene cluster, and to knock-in a green fluorescent protein expression cassette employing a cloning-independent methodol., without introducing undesirable redundant sequences at the modified locus in the B. subtilis 1A751. Also, the mini-mazF-cassette could be used repeatedly to delete multiple genes or gene clusters with only a 2- to 2.5-kb PCR-fused fragment, which largely reduced the frequency of nucleic acid mutations generated by PCR compared to previous reports. We further demonstrated that the frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%. The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3 days. This modified cassette has remarkable improvement compared to existing approaches and is applicable for available manipulating Bacillus species chromosomes.
- 24Datsenko, K. A., Wanner, B. L., and Beckwith, J. (2000) One-Step Inactivation of Chromosomal Genes in Escherichia Coli K-12 Using PCR Products. Proc. Natl. Acad. Sci. U. S. A. 97 (12), 6640– 6645, DOI: 10.1073/pnas.12016329724https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXktFais7c%253D&md5=85388def19d7c14ddada4b70a0bec1eeOne-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR productsDatsenko, Kirill A.; Wanner, Barry L.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (12), 6640-6645CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The authors have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homol. to the targeted gene(s). In this procedure, recombination requires the phage λ Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy no. plasmid. To demonstrate the utility of this approach, the authors generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the resp. PCR products, the authors made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, esp. in genome anal. of E. coli and other bacteria because the procedure can be done in wild-type cells.
- 25Li, Y., Lin, Z., Huang, C., Zhang, Y., Wang, Z., Tang, Y., Chen, T., and Zhao, X. (2015) Metabolic Engineering of Escherichia Coli Using CRISPR–Cas9Meditated Genome Editing. Metab. Eng. 31, 13– 21, DOI: 10.1016/j.ymben.2015.06.00625https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFaiurjM&md5=1285f024ef6155504519f691e0d83edfMetabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editingLi, Yifan; Lin, Zhenquan; Huang, Can; Zhang, Yan; Wang, Zhiwen; Tang, Ya-jie; Chen, Tao; Zhao, XuemingMetabolic Engineering (2015), 31 (), 13-21CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)Engineering cellular metab. for improved prodn. of valuable chems. requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli. This system enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously. We also found that cells with intact mismatch repair system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential, we used our method to integrate the β-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate (MEP) pathway and central metabolic pathways for β-carotene overprodn. We collectively tested 33 genomic modifications and constructed more than 100 genetic variants for combinatorially exploring the metabolic landscape. Our best producer contained 15 targeted mutations and produced 2.0 g/L β-carotene in fed-batch fermn.
- 26Koo, B. M., Kritikos, G., Farelli, J. D., Todor, H., Tong, K., Kimsey, H., Wapinski, I., Galardini, M., Cabal, A., Peters, J. M. (2017) Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus Subtilis. Cell Syst. 22, 291– 305, DOI: 10.1016/j.cels.2016.12.013There is no corresponding record for this reference.
- 27Altenbuchner, J. (2016) Editing of the Bacillus Subtilis Genome by the CRISPR-Cas9 System. Appl. Environ. Microbiol. 82 (17), 5421– 5427, DOI: 10.1128/AEM.01453-1627https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVahurvJ&md5=ede5e7d6c146a01bbac9c39f4872a929Editing of the Bacillus subtilis genome by the CRISPR-Cas9 systemAltenbuchner, JosefApplied and Environmental Microbiology (2016), 82 (17), 5421-5427CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)The clustered regularly interspaced short palindromic repeat (CRISPR)-assocd. (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temp.-sensitive replication origin of plasmid pE194ts for B.subtilis, and a kanamycin resistance gene working in both organisms. This sgRNA guides the Cas9 nuclease to its target. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Repair of the DSBs and the required modification of the genome are achieved by adding homol. templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homol. templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B.subtilis chromosome and by repair of the trpC2 mutation of B.subtilis 168. For genome editing, it carries the cas9 gene under the control of the B.subtilis mannose-inducible promoter PmanP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. The 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site.
- 28Veening, J.-W., Murray, H., and Errington, J. (2009) A Mechanism for Cell Cycle Regulation of Sporulation Initiation in Bacillus Subtilis. Genes Dev. 23, 1959– 1970, DOI: 10.1101/gad.52820928https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVegurbK&md5=0b50b569cd9816014dcfd87e2d730258A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilisVeening, Jan-Willem; Murray, Heath; Errington, JeffGenes & Development (2009), 23 (16), 1959-1970CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)Coordination of DNA replication with cellular development is a crucial problem in most living organisms. Bacillus subtilis cells switch from vegetative growth to sporulation when starved. Sporulation normally occurs in cells that have stopped replicating DNA and have two completed chromosomes, one destined for the prespore and the other for the mother cell. It has long been recognized that there is a sensitive period in the cell cycle during which the initiation of spore development can be triggered, presumably to allow for the generation of exactly two complete chromosomes. However, the mechanism responsible for this has remained unclear. Here we show that the sda gene, previously identified as a checkpoint factor preventing sporulation in response to DNA damage, exerts cell cycle control over the initiation of sporulation. Expression of sda occurs in a pulsatile manner, with a burst of expression each cell cycle at the onset of DNA replication. Up-regulation of the intrinsically unstable Sda protein, which is dependent on the active form of the DNA replication initiator protein, DnaA, transiently inhibits the initiation of sporulation. This regulation avoids the generation of spore formers with replicating chromosomes, which would result in diploid or polyploid spores that we show have reduced viability.
- 29Kobayashi, K., Ehrlich, S. D., Albertini, A., Amati, G., Andersen, K. K., Arnaud, M., Asai, K., Ashikaga, S., Aymerich, S., Bessieres, P. (2003) Essential Bacillus Subtilis Genes. Proc. Natl. Acad. Sci. U. S. A. 100 (8), 4678– 4683, DOI: 10.1073/pnas.073051510029https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjt12ns7o%253D&md5=8c03de4a0dd3ddf96ae5575d372346b6Essential bacillus subtilis genesKobayashi, K.; Ehrlich, S. D.; Albertini, A.; Amati, G.; Andersen, K. K.; Arnaud, M.; Asai, K.; Ashikaga, S.; Aymerich, S.; Bessieres, P.; Boland, F.; Brignell, S. C.; Bron, S.; Bunai, K.; Chapuis, J.; Christiansen, L. C.; Danchin, A.; Debarbouille, M.; Dervyn, E.; Deuerling, E.; Devine, K.; Devine, S. K.; Dreesen, O.; Errington, J.; Fillinger, S.; Foster, S. J.; Fujita, Y.; Galizzi, A.; Gardan, R.; Eschevins, C.; Fukushima, T.; Haga, K.; Harwood, C. R.; Hecker, M.; Hosoya, D.; Hullo, M. F.; Kakeshita, H.; Karamata, D.; Kasahara, Y.; Kawamura, F.; Koga, K.; Koski, P.; Kuwana, R.; Imamura, D.; Ishimaru, M.; Ishikawa, S.; Ishio, I.; Le Coq, D.; Masson, A.; Mauel, C.; Meima, R.; Mellado, R. P.; Moir, A.; Moriya, S.; Nagakawa, E.; Nanamiya, H.; Nakai, S.; Nygaard, P.; Ogura, M.; Ohanan, T.; O'Reilly, M.; O'Rourke, M.; Pragai, Z.; Pooley, H. M.; Rapoport, G.; Rawlins, J. P.; Rivas, L. A.; Rivolta, C.; Sadaie, A.; Sadaie, Y.; Sarvas, M.; Sato, T.; Saxild, H. H.; Scanlan, E.; Schumann, W.; Seegers, J. F. M. L.; Sekiguchi, J.; Sekowska, A.; Seror, S. J.; Simon, M.; Stragier, P.; Studer, R.; Takamatsu, H.; Tanaka, T.; Takeuchi, M.; Thomaides, H. B.; Vagner, V.; van Dijl, J. M.; Watabe, K.; Wipat, A.; Yamamoto, H.; Yamamoto, M.; Yamamoto, Y.; Yamane, K.; Yata, K.; Yoshida, K.; Yoshikawa, H.; Zuber, U.; Ogasawara, N.Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (8), 4678-4683CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)To est. the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ≈4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metab., with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the detn. of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.
- 30Juhas, M., Reuß, D. R., Zhu, B., and Commichau, F. M. (2014) Bacillus Subtilis and Escherichia Coli Essential Genes and Minimal Cell Factories after One Decade of Genome Engineering. Microbiology (London, U. K.) 160, 2341– 2351, DOI: 10.1099/mic.0.079376-030https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXktVyjuw%253D%253D&md5=041b4443f4ab77c644edeed44e58036eBacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineeringJuhas, Mario; Reuss, Daniel R.; Zhu, Bingyao; Commichau, Fabian M.Microbiology (Reading, United Kingdom) (2014), 160 (11), 2341-2351CODEN: MROBEO; ISSN:1350-0872. (Society for General Microbiology)A review. Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell "chassis". Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnol. applications. Detn. of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome redn. analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.
- 31Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., and Yang, S. (2015) Multigene Editing in the Escherichia Coli Genome via the CRISPR-Cas9 System. Appl. Environ. Microbiol. 81 (7), 2506– 2514, DOI: 10.1128/AEM.04023-1431https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvV2hsbY%253D&md5=7738bd7921f45b04f0f3dcac4d99a4d8Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 systemJiang, Yu; Chen, Biao; Duan, Chunlan; Sun, Bingbing; Yang, Junjie; Yang, ShengApplied and Environmental Microbiology (2015), 81 (7), 2506-2514CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.
- 32Sung, W., Ackerman, M. S., Gout, J.-F., Miller, S. F., Williams, E., Foster, P. L., and Lynch, M. (2015) Asymmetric Context-Dependent Mutation Patterns Revealed through Mutation-Accumulation Experiments. Mol. Biol. Evol. 32 (7), 1672– 1683, DOI: 10.1093/molbev/msv05532https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs12rsL%252FP&md5=56462d5a5628bf8c6550e9b490aa0b25Asymmetric context-dependent mutation patterns revealed through mutation-accumulation experimentsSung, Way; Ackerman, Matthew S.; Gout, Jean-Francois; Miller, Samuel F.; Williams, Emily; Foster, Patricia L.; Lynch, MichaelMolecular Biology and Evolution (2015), 32 (7), 1672-1683CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)Despite the general assumption that site-specific mutation rates are independent of the local sequence context, a growing body of evidence suggests otherwise. To further examine context-dependent patterns of mutation, we amassed 5,645 spontaneous mutations in wild- type (WT) and mismatch-repair deficient (MMR-) mutation-accumulation (MA) lines of the gram-pos. model organism Bacillus subtilis. We then analyzed >7,500 spontaneous base-substitution mutations across B. subtilis, Escherichia coli, and Mesoplasma florum WT and MMR- MA lines, finding a context-dependent mutation pattern that is asym. around the origin of replication. Different neighboring nucleotides can alter site-specific mutation rates by as much as 75-fold, with sites neighboring G:C base pairs or dimers involving alternating pyrimidine-purine and purine-pyrimidine nucleotides having significantly elevated mutation rates. The influence of context-dependent mutation on genome architecture is strongest in M. florum, consistent with the reduced efficiency of selection in organisms with low effective population size. If not properly accounted for, the disparities arising from patterns of context-dependent mutation can significantly influence interpretations of pos. and purifying selection.
- 33Lee, H., Popodi, E., Tang, H., and Foster, P. L. (2012) Rate and Molecular Spectrum of Spontaneous Mutations in the Bacterium Escherichia Coli as Determined by Whole-Genome Sequencing. Proc. Natl. Acad. Sci. U. S. A. 109 (41), E2774– E2783, DOI: 10.1073/pnas.121030910933https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFKntbjM&md5=e44fb70553c7eadc11516ee3346dcd4dRate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencingLee, Heewook; Popodi, Ellen; Tang, Haixu; Foster, Patricia L.Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (41), E2774-E2783, SE2774/1-SE2774/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and mol. processes. In this report, we analyze spontaneous mutations accumulated over thousands of generations by wild-type Escherichia coli and a deriv. defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. col strain is ∼1 × 10-3 per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5'ApC3'/3'TpG5' sites (where bases 5'A and 3'T are mutated) and, to a lesser extent, at 5'GpC3'/3'CpG5' sites (where bases 5'G and 3'C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that det. mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions.
- 34Wang, K., de la Torre, D., Robertson, W. E., and Chin, J. W. (2019) Programmed Chromosome Fission and Fusion Enable Precise Large-Scale Genome Rearrangement and Assembly. Science (Washington, DC, U. S.) 365, 922– 926, DOI: 10.1126/science.aay073734https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslSqsbvO&md5=aa1e8a441a5d31f7da1c52fc51864646Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assemblyWang, Kaihang; de la Torre, Daniel; Robertson, Wesley E.; Chin, Jason W.Science (Washington, DC, United States) (2019), 365 (6456), 922-926CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The model bacterium Escherichia coli has a single circular chromosome. Wang et al. created a method to fragment the E. coli genome into independent chromosomes that can be modified, rearranged, and recombined. The efficient fission of the unmodified E. coli genome into two defined, stable pairs of synthetic chromosomes provides common intermediates for large-scale genome manipulations such as inversion and translocation. Fusion of synthetic chromosomes from distinct cells generated a single genome in a target cell. Precise, rapid, large-scale genome engineering operations are useful tools for creating diverse synthetic genomes.
- 35Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A. (2019) Search-and-Replace Genome Editing without Double-Strand Breaks or Donor DNA. Nature (London, U. K.) 576, 149– 157, DOI: 10.1038/s41586-019-1711-435https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFGns7rO&md5=1fbd690d78a81c3b90ad5f9dad5be086Search-and-replace genome editing without double-strand breaks or donor DNAAnzalone, Andrew V.; Randolph, Peyton B.; Davis, Jessie R.; Sousa, Alexander A.; Koblan, Luke W.; Levy, Jonathan M.; Chen, Peter J.; Wilson, Christopher; Newby, Gregory A.; Raguram, Aditya; Liu, David R.Nature (London, United Kingdom) (2019), 576 (7785), 149-157CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homol.-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants assocd. with human diseases.
- 36Velázquez, E., Lorenzo, V. de, and Al-Ramahi, Y. (2019) Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery. ACS Synth. Biol. 8 (9), 2186– 2193, DOI: 10.1021/acssynbio.9b0029336https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsF2ntbzN&md5=f06edd98e918993b500d50caa1978a9cRecombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron DeliveryVelazquez, Elena; Lorenzo, Victor de; Al-Ramahi, YamalACS Synthetic Biology (2019), 8 (9), 2186-2193CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Group II introns were developed time ago as tools for the construction of knockout mutants in a wide range of organisms, ranging from Gram-pos. and Gram-neg. bacteria to human cells. Utilizing these introns is advantageous because they are independent of the host's DNA recombination machinery, they can carry heterologous sequences (and thus be used as vehicles for gene delivery), and they can be easily retargeted for subsequent insertions of addnl. genes at the user's will. Alas, the use of this platform has been limited, as insertion efficiencies greatly change depending on the target sites and cannot be predicted a priori. Moreover, the ability of introns to perform their own splicing and integration is compromised when they carry foreign sequences. To overcome these limitations, we merged the group II intron-based TargeTron system with CRISPR/Cas9 counter-selection. To this end, we first engineered a new group-II intron by replacing the retrotransposition-activated selectable marker (RAM) with ura3 and retargeting it to a new site in the lacZ gene of E. coli. Then, we showed proved that directing CRISPR/Cas9 towards the wild-type sequences dramatically increased the chances of finding clones that integrated the retrointron into the target lacZ sequence. The CRISPR-Cas9 counter selection strategy presented herein thus overcomes a major limitation that has prevented the use of group II introns as devices for gene delivery and genome editing at large in a recombination-independent fashion.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.9b00400.
Supplementary text explains barcode design; Supplementary figures of plasmid maps used for barcoding and description of barcode stability assays results; Supplementary tables show the efficiency of the barcoding methods and the mutation rate comparison with bibliography (PDF)
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