In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA–CRISPR-Cas12 Assay

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. “One-pot” assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT–CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT–CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA–Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.


Technology Comparison
Table S1.A collection of NAAT-CRISPR-Cas one-pot diagnostics assays that increase sensitivity by mitigating the competition between amplification and detection.
The pH was then adjusted to 8.5 with potassium acetate.

Preparation of SPR running buffer
Due to the large volumes necessary for SPR experiments, an SPR running buffer was created by adding the following reagents to a final concentration of 25mM Tris, 100mM Potassium acetate, 2mM Dithiothreitol, and 20mM Magnesium acetate. 13Nuclease-free water (Thermo Fischer Scientific, Waltham, USA) was added and the pH adjusted to 7.9 using potassium hydroxide.

Preparation of RPA reaction buffer
The RPA reaction buffer was the SPR running buffer with the addition of 5% PEG to mimic the RPA reaction mixture. 13

Generation of target DNA
Target DNA was produced by combining the following reagents (as final concentrations) as follows: ThermoScientific DreamTaq Hot Start PCR Master Mix (2X) 1x (Thermo Fischer Scientific, Waltham, USA), HPV16 RPA Primer Forward 500 nM, HPV16 RPA Primer Reverse 500 nM, template DNA 250 aM and nucleasefree water.The thermal routine utilized was as follows: initial denaturation of 95°C for 2 minutes, then 40 cycles of denaturation (95°C for 30 seconds), annealing (62.3°C for 30 seconds), and elongation (72°C for 1 minute).A final elongation step of 72°C for 1 minute was performed.

PCR Evaluation of Clinical Samples
Clinical samples were collected by a gynecologist using a Viba brush (Rovers Medical Devices, Oss, Netherlands).The cervix and the superficial vaginal canal were swabbed with the brush, which then was rinsed in Hologic ThinPrep medium (Hologic, Mississauga, Canada).DNA was extracted from the ThinPrep medium using a STARMAG 96 x 4 Universal Cartridge Kit (Seegene, Seoul, Republic of Korea), and analyzed using the Allplex HPV28 and Anyplex HPV HR detection assays on a Microlab STAR (Hamilton, Reno, USA) device equipped with a thermal cycler.
Table S3.Analysis of patient-derived samples using Allplex and Anyplex.

Conjugation of Cas12 with Alexa Fluor 488
The protein was fluorescently labelled using a Lightning-Link Alexa Fluor 488 Fast Conjugation Kit (Abcam, Cambridge, U.K.), following the manufacturer's instructions.In brief, LbCas12a was diluted with a high-salt buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.5) to yield a final concentration of 1 mg mL -1 .Then, 100 µL of the protein dilution was added to the conjugation kit and the mixture incubated for 15 min at room temperature.The reaction was stopped by the addition of a quencher.

Plotting of Figure 2
Plots (a) and (b) in Figure 2 show a mean line (linearly interpolated between data points) with the shaded error range indicating ±3 standard deviations.Although the reading was not continuous (measurement every 2 minutes) the data is represented in this way for clarity.Plot (c) in Figure 2 shows all data points found (3 per concentration per assay format).The TTR was found as described below in the section "Time-to-result determination".

Time-to-result determination
The time-to-result algorithm was based on the work of Pena et al,. 14which states that the "The maximal slope value for the negative samples in an experiment was determined, and the SD of the slopes for the negative samples was calculated.The maximal slope value plus three SD values calculated from the negative samples was set as the cutoff value.The TTR (in minutes) was defined as the time at which the fluorescence of a sample surpassed the cutoff value in three consecutive recordings."

Reaction modelling
To deduce reasonable values for kamp, we modelled the amplification rates alone, without the influence of Casmediated processes.6][17] These times were characterized by when the amplification reaction is able to move from its initiation phase to its exponential phase (Figure S1).

Diffusion model -sgRNA and Cas12a
Utilizing a version of the Smoluchowski derivation of the Arrhenius equation (Equation S1), 18 we solved for the percentage of Cas12 and sgRNA experiencing a collision with each other across critical early-reaction times (Figure S2).
Terms specific to Equation S1 include , ,  ' , [A] and [B] which represent the cross-sectional area of molecule A, the unitless fraction of the area of molecule B, the summed diffusion coefficient of molecules A and B, the concentration of molecule A in the reaction, and the concentration of molecule B in the reaction, respectively.Molecule A was chosen to be Cas12a.The radius of Cas12a used (to calculate the area "A") was taken as 3.7 nm as described by Bonini. 19The fraction of molecule B (sgRNA) that is able to bind to molecule A was assumed to be 0.51.This fraction represents the portion of the sgRNA that forms the repeat or scaffold, the portion of the sgRNA that is involved in binding to the Cas12a. 10" ' " was the summation of diffusion coefficients from FCS, found to be 16.7 ± 2.14 µm 2 /s and 47.02 ± 2.7 µm 2 /s, for the Cas12a and sgRNA, respectively.The shaded region in plot a) indicates the error found in FCS (diffusion coefficient error for Cas12a = ± 2.14 µm 2 /s and diffusion coefficient error for the sgRNA = ± 2.7 µm 2 /s) propagated through the " ' " term in accordance with the standard propagation of error equation, below.
!,  ) , and  !,) are defined as the standard deviation of error of the diffusion coefficient of molecule A, molecule B, and the covariance of molecules A & B, respectively.The covariance term was assumed to be zero for the effect of one particle's diffusion coefficient on the other when unbound.
A key assumption in this discrete solution is that in each instance of a Cas12a and sgRNA collision, they are assumed to have bound, focusing the investigation on diffusion rather than orientation and collision energy.Data S8 indicate minimal delay before the reaction reaches its final concentration of RNP; this suggests that complex formation is not diffusion-controlled.The code from this analysis is available at: https://github.com/hbdadboy/In-situ-Complexation-Improves-One-Pot.

Establishing a detection threshold
To understand what constitutes a detectable concentration of target in our model, a detection threshold was established on the plate reader where the data for Figures 2 and 3 were collected.A reaction was created with the exact reagents and final concentrations as the precomplexed reaction above, with the following modifications: omission of target DNA and inclusion of cleaved fluorescent reporter (SI Table 1).The standard (uncleaved) fluorescence reporter was included in the solution as a control.The cleaved fluorescent reporter was titrated into reaction mixtures at final concentrations of 100 nM, 80 nM, 60 nM, 40 nM, 20 nM and 0 nM.From these concentrations, a line of best fit was constructed.The detection threshold was determined as the intersection of this line with the 99.7% confidence interval set around the mean of the zero value (0 nM of cleaved reporter).

Figure S1 .
Figure S1.Modelling amplification times alone (without the addition of Cas proteins) to find reasonable doubling times.

Figure S2 .
Figure S2.a) Modelling diffusion times according to the Smoluchowski derivation of the Arrhenius equation, as described above.b) FCS curve fits to determine diffusion coefficients.Normalized autocorrelation curves for both the sgRNA and Cas12a are overlaid with theoretical FCS curve fit.c) Residuals obtained from the curve fit for both the components, demonstrating the suitability of the model.

Table S2 .
Sequences of the oligonucleotides used in this study.All oligonucleotides were commercially produced by Microsynth AG, Switzerland.