Methods

Fundamental limits of amplification-free CRISPR-Cas12 and Cas13 diagnostics

Enzyme preparation

LbCas12a was purchased from New England Biolabs (NEB, MA, USA) at a concentration of 100 μM. AsCas12a and LwaCas13a were obtained from IDT at concentrations, respectively, of 64 and 72 μM. The plasmids for AapCas12b (pAG001 His6-TwinStrep-SUMO-AapCas12b) were a gift from Omar Abudayyeh and Jonathan Gootenberg (http://n2t.net/addgene:153162; RRID: Addgene_153162). The plasmids for LbuCas13a (pC0072 LbuCas13a His6-TwinStrep-SUMO-BsaI) were a gift from Feng Zhang (http://n2t.net/addgene:115267; RRID: Addgene_115267). Expression plasmids were transformed into Rosetta 2(DE3). pLysS competent cells (Millipore Corporation, MA, USA) and protein expression was performed in 2 L of Terrific Broth supplemented with 50 μg mL-1 carbenicillin (Teknova Inc., CA, USA) at 18 °C for 16 hours. Proteins were purified according to the published protocol26 without major modification except the harvested cells were lysed by sonication. Proteins were stored in Storage Buffer (600 mM NaCl, 50 mM Tris-HCl pH 7.5, 5% glycerol, 2mM DTT) at −80 °C in 10 μl aliquots. After purification, the stock AapCas12b and LbuCas13a enzyme concentrations were respectively 4.4 and 39 μM.

Trans-cleavage kinetics experiments

To measure the trans-cleavage kinetics of Cas12 enzymes, we first prepared 1 μM solutions of the Cas12-gRNA complex. This was achieved by incubating a mixture of 1 μM synthetic gRNA with at least 2-fold excess of the corresponding Cas12 at 37 °C for 30 min on a hot plate. Cas12-gRNA complexes were then activated for trans-cleavage activity by mixing and incubating these complexes with synthetic ss- or dsDNA at 100 nM concentration at 37 °C for 30 min on a hot plate. The latter step yielded a solution with an activated Cas12 concentration of 100 nM. We performed the trans-cleavage kinetics assay using 1 nM of activated Cas12 and varied ssDNA reporter concentrations of 15.63, 31.25, 62.5, 125, 250, 500, 1000, and 2000 nM. Three replicates were taken for each concentration. All LbCas12a and AsCas12a reactions were buffered in 1x NEBuffer 2.1 (composed of 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 100 μg mL-1 bovine serum albumin at pH = 7.9) and all AapCas12b reactions were buffered in 1x isothermal amplification buffer (composed of 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween® 20 at pH 8.8) supplemented with 10 mM MgCl2.17 Most Cas12 trans-cleavage reactions were run at 37 °C (AapCas12b was reacted at 37 and 60 °C). Cas12 kinetics data was measured using a MiniOpticon thermal cycler (Bio-Rad Laboratories, CA, USA), and fluorescence was measured every 30 s. A fluorescence calibration curve (Fig. S1) was used to convert fluorescence values (in arbitrary units) to molar concentrations (in nM). The initial reaction velocities (in nM s-1) for Michaelis-Menten analyses were obtained using a linear fit to the first ~300 s of the reaction progress curves. The reaction velocities versus reporter concentration data were fitted to the Michaelis-Menten equation using GraphPad Prism 9 (GraphPad Software, CA, USA) to obtain kcat and KM.

The Cas13 trans-cleavage assay was like that followed for Cas12 with the following differences. First, Cas13-gRNA was activated using synthetic ssRNA targets. The reporter ssRNA concentrations used were 125, 250, 500, 1000, 2000, 4000, and 6000 nM. The buffer used for all Cas13 reactions consisted of 20 mM HEPES, 50 mM NaCl, 10 mM MgCl2, RNase inhibitor 1 U mL-1, and 5% glycerol at pH ≈ 6.8. Cas13 kinetics data was measured using an Infinite 200 PRO plate reader (Männedorf, Switzerland), and fluorescence was measured every 30 s. Due to the necessary high reporter concentrations, an inner filter effect was observed which reduced the apparent fluorescence of reporters. A power law relation was therefore used to convert fluorescence values to molar concentrations (Fig. S10).21 The initial reaction velocities (in nM s-1) for Michaelis-Menten analyses were obtained using a linear fit to the first ~200 s of the reaction progress curves.

Limit of detection experiments

To determine the LoD for each gRNA-target pair for both Cas12 and Cas13 enzymes, we first prepared a solution containing 1 μM Cas-gRNA complex using the procedure described for the trans-cleavage assay. Later, individual reactions were set up by mixing Cas-gRNA complex, reporter ssDNA or ssRNA, and varying amounts of target activator (ssDNA/dsDNA/RNA), for each enzyme in the corresponding reaction buffer. For this, we used 100 nM of Cas-gRNA, 200 nM and 1 μM reporters for Cas12 and Cas13 experiments respectively and varied the target activator concentration between 10 nM and 10 fM in 10-fold dilutions. Fluorescence readouts were taken in 1 min intervals for a total of 60 min on the ABI 7500 Fast thermal cycler (Applied Biosystems, CA, USA). LoDs were estimated using two methods: endpoint fluorescence readout and maximum reaction velocity (i.e., slope). For the former method, we used the fluorescence readout at 60 min as the measure of signal. For the maximum reaction velocity method, we used the maximum value of instantaneous reaction velocity over the course of the experiment (obtained from the slope of fluorescence versus time, averaged over 3 min) as a measure of signal. A 4PL curve was then fit to the data for each enzyme and method.

Oligo preparation

ssDNA and RNA oligonucleotides (target activator, gRNA, and reporter) were purchased from Integrated DNA Technologies (IDT, IA, USA), Elim Biopharmaceuticals Inc. (CA, USA), and GeneLink Inc. (FL, USA). RNA oligonucleotides (from IDT and GeneLink) were resuspended to 100 μM in RNA reconstitution buffer (GeneLink). DNA oligonucleotides (Elim) were obtained at 100 μM concentration in nuclease-free water. The complete list of oligos used in this study is provided (Tables S2-S4). For Cas12 experiments, synthetic dsDNA target oligos were prepared by hybridizing 10 μM of ssDNA template with 50 μM of complementary ssDNA in 1x NEBuffer 2.1 (NEB), to provide a solution with an effective dsDNA concentration of 10 μM. The hybridization protocol included a 95 °C hold for 2 min, followed by a slow cool (0.1 °C s-1) to 25 °C. The protocol was carried out using the MiniOpticon thermal cycler (Bio-Rad).

Calculation of LoD

This section describes the calculation of the endpoint-based (LE) and velocity-based (LV) LoDs. Specifically, LE was quantified as the molar concentration of the target at which the measured endpoint fluorescence signal equaled the endpoint threshold. The endpoint threshold TE was defined as where μE,NTC and σE,NTC respectively correspond to the mean and standard deviation of the endpoint fluorescence measurements of the NTC samples. Analogously, LV was based on the maximum observed reaction velocity and its comparison to the background, non-specific activity of the enzyme complex with fluorescence probes and/or probe degradation (i.e., the NTC cleavage activity). The analogous, velocity-based threshold TV was defined as where μV,NTC and σV,NTC respectively correspond to the mean and standard deviation of the maximum cleavage (measured) velocities of the NTC samples.

Article TitleFundamental limits of amplification-free CRISPR-Cas12 and Cas13 diagnostics

Abstract

Interest in CRISPR diagnostics continues to increase. CRISPR-Cas12 and -Cas13 based detection are particularly interesting as they enable highly specific detection of nucleic acids. The fundamental sensitivity limits of Cas12 and Cas13 enzymes are governed by their kinetic rates and are critical to develop amplification-free assays. However, these kinetic rates remain poorly understood and their reporting has been inconsistent. We here measure kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a and LbuCas13a) and evaluate their limits of detection (LoD) for amplification-free target detection. Collectively, we here present quantitation of enzyme kinetics for 14 gRNAs and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. Importantly, we also validate the self-consistency our measurements by comparing trends and limiting behaviors with a Michaelis-Menten, trans-cleavage reaction kinetics model. Our measurements reveal that activated Cas12 and Cas13 enzymes exhibit typical trans-cleavage catalytic efficiencies between order 105 and 106 M-1 s-1. Moreover, for assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, we find most CRISPR enzymes have an amplification-free LoD in the picomolar range. We find also that successful detection of target requires cleavage (by activated CRISPR enzyme) of at least ~0.1% of the fluorescent reporter molecules. This fraction of cleaved reporters is required to differentiate signal from background, and we hypothesize that this fraction is largely independent of the detection method (i.e., endpoint vs reaction velocity). Our results provide a map of the feasible application range and highlight areas of improvement for the emerging field of CRISPR diagnostics.


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