A chemical-enhanced system for CRISPR-Based nucleic acid detection

Abstract

The CRISPR-based nucleic acid detection systems have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance, and reveal a crucial role of bovine serum albumin in enhancing trans-cleavage activity of Cas12a/Cas13a effectors. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness, and helps to develop enhanced reagent formula or test kit towards practical application of CRISPR-based diagnostics.

Keywords: COVID-19; CRISPR; Chemical; Detection; Diagnostics; SARS-CoV-2.

Fig. 1

Optimization of key parameters for CRISPR detection systems. (A) Schematic description of CRISPR-Cas12a/Cas13a detection systems for nucleic acids. (B, C) Comparison of the indicated commercial reaction buffers for their effects on AsCas12a- and LwaCas13a-mediated trans-cleavage activity. A synthetic DNA template corresponding to SARS-CoV-2 N gene (B), and a synthetic RNA template corresponding to SARS-CoV-2 S gene (C) are used. (D, E) Evaluation of the indicated molecular ratio of Cas protein:crRNA for the effect on AsCas12a detection (D) and LwaCas13a detection (E). (F– I) The effect of indicated amount of fluorescence reporter on CRISPR detection assays using either AsCas12a (F and G) or LwaCas13a (H and I). The results are shown by either real-time recording of fluorescence signal (F and H) or endpoint (60 min) visualization (G and I) under blue light illuminator. R1, R2 and R3 indicate three replicates. Error bars represent mean ± s.d. (n = 3). a. u., arbitrary unit. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Dynamic detection range of target amount (A, B) Evaluation of the detection sensitivity of AsCas12a (A) and LwaCas13a (B) systems with indicated amount of synthetic templates. (C, D) AsCas12a-mediated detection power for indicated amount of pure targets using plasmid bearing SARS-CoV-2 N gene fragment. The fluorescence signals are shown by either real-time recording of fluorescence signal (C) or endpoint (60 min) visualization (D) under blue light illuminator. R1, R2 and R3 indicate three replicates. (E, F) LwaCas13a-mediated detection power for indicated amount of pure targets using in vitro transcribed RNAs corresponding to SARS-CoV-2 N gene fragment. Error bars represent mean ± s.d. (n = 3). a. u., arbitrary unit. Unpaired two-tailed t-test, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns means not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Effects of chemical additives on CRISPR detection phase (A) Schematic diagram showing the workflow to evaluate chemical additive effect on CRISPR detection system. (B, C) Endpoint (60 min) recording of fluorescence detection signals for either AsCas12a (B) or LwaCas13a (C) system. w /o template means no input; non-target means non-targeting template (N gene template as S gene non-target, and vice versa); w/o chemical means no chemical addition. (D) Comparison of fluorescent signals resulted from AsCas12a-based detection with or without L-proline addition. Two-way ANOVA test, ****p < 0.0001; ns means not significant. (E) The effect of BSA addition on AsCas12a-based detection system. Two-way ANOVA test, ****p < 0.0001. (F) Comparison of different concentrations of BSA addition for the effects on AsCas12a-mediated detection. Unpaired two-tailed t-test, ***p < 0.001, ****p < 0.0001. (G) Evaluation of L-proline's effect on protecting BSA from heat-induced denature and BSA's capability to enhance AsCas12a detection. BSA along or BSA co-incubated with L-proline are treated at 70 °C for 15 min or 30 min, before serving as additive in AsCas12a assays. Two-way ANOVA test, ****p < 0.0001. Error bars represent mean ± s.d. (n = 3). a. u., arbitrary unit.

Fig. 4

Effects of chemical additives on target amplification phase (A) Schematic diagram showing the workflow to evaluate LAMP and RPA detection for target RNAs. (B) Evaluation of different chemical additives for the effect on specific target amplification as determined by AsCas12a detection using purified LAMP products. LAMP is performed for 15 min at 62 °C. Endpoint (60 min) recording of fluorescence detection signals are shown. CRISPR Ctrl indicates no nucleic acid input for AsCas12a detection; w/o template means no input for LAMP reaction. (C) The fluorescence signal dynamics is shown for the selected samples in (B). (D–G) Comparison of L-proline addition in LAMP reaction phase determined by AsCas12a. The template and primer set for this LAMP reaction is quoted from Broughton et al. (N #1). LAMP is performed for 30 min at 62 °C. Purified LAMP products (D and E) or after proper dilution (F and G) are subjected to AsCas12a detection. (H–K) Similar assays are performed using different template and primer set for LAMP reaction according to Joung et al. (N #2). (L) qRT-PCR quantification of varied SARS-CoV-2 reference RNA. (M) Effect of L-proline on AsCas12a detection with varied amount of template. (N) Correlation of Ct values and enhancement fold change of AsCas12a detection when adding L-proline. Two-way ANOVA test, ****p < 0.0001. ns means not significant. Error bars represent mean ± s.d. (n = 3). a. u., arbitrary unit.

Fig. 5

CECRID deployment for detection of SARS-CoV-2 pseudovirus (A) Schematic diagram showing the principle and application workflow of CECRID systems. (B– D) Comparative analysis of SARS-CoV-2 N gene-bearing pseudovirus detection with CECRID and normal detection platforms. The template and primer set for this LAMP reaction is N#1. Various amount of pseudoviral particles in VTM are collected and viral RNA is purified by commercial RNA purification kit. RT-LAMP is performed with viral RNA at 62 °C for 30 min followed by 20 min of 80 °C inactivation. The purified amplification products (1:50 dilution) are then subjected to AsCas12a-based fluorescence and lateral flow strip detection. For CECRID, L-proline is added in both the LAMP phase and CRISPR detection phase. The endpoint (60 min) fluorescence signal is shown by either bar plot (B), direct visualization (C) or lateral flow strip using biotin-labeled reporter (D). The significant band in test line represents positive result. C: control line. T: test line. (E–G) Similar assays are performed using an independent pseudovirus/template and primer set N #2. Two-way ANOVA test, ****p < 0.0001. ns means not significant. Error bars represent mean ± s.d. (n = 3). a. u., arbitrary unit.