Nucleic acid detection with CRISPR-Cas13a/C2c2

Abstract

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.

Figure 1. SHERLOCK is capable of single-molecule nucleic acid detection

(A) Schematic of SHERLOCK. (B) Schematic of ssRNA target detected via the Cas13a collateral detection. The target site is highlighted in blue. (C) Cas13a detection of RNA with RPA amplification (SHERLOCK) can detect ssRNA target at concentrations down to ~2 aM, more sensitive than Cas13a alone. (n=4 technical replicates; bars represent mean ± s.e.m.) (D) SHERLOCK is also capable of single-molecule DNA detection. (n=4 technical replicates; bars represent mean ± s.e.m.)

Figure 2. Cas13a detection can be used to sense viral and bacterial pathogens

(A) Schematic of ZIKV RNA detection by SHERLOCK. (B) SHERLOCK is capable of highly sensitive detection of the ZIKV lentiviral particles. (n=4 technical replicates, two-tailed Student t-test; ****, p < 0.0001; bars represent mean ± s.e.m.; n.d., not detected) (C) Schematic of ZIKV RNA detection with freeze-dried Cas13a on paper (D) Paper-based SHERLOCK is capable of highly sensitive detection of ZIKV lentiviral particles. (n=4 technical replicates, two-tailed Student t-test; **, p < 0.01; ****, p < 0.0001; bars represent mean ± s.e.m.) (E) Schematic of SHERLOCK detection of ZIKV RNA isolated from human clinical samples. (F) SHERLOCK is capable of highly sensitive detection of human ZIKV-positive serum (S) or urine (U) samples. Approximate concentrations of ZIKV RNA shown were determined by qPCR. (n=4 technical replicates, two-tailed Student t-test; ****, p < 0.0001; bars represent mean ± s.e.m.; n.d., not detected) (G) Schematic of using SHERLOCK to distinguish bacterial strains using a universal 16S rRNA gene V3 RPA primer set. (H) SHERLOCK achieves sensitive and specific detection of E. coli or P. aeruginosa gDNA. (n=4 technical replicates, two-tailed Student t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; bars represent mean ± s.e.m.). Ec, Escherichia coli; Kp, Klebsiella pneumoniae; Pa, Pseudomonas aeruginosa; Mt, Mycobacterium tuberculosis; Sa, Staphylococcus aureus.

Figure 3. Cas13a detection can discriminate between similar viral strains

(A) Schematic of ZIKV strain target regions and the crRNA sequences used for detection. SNPs in the target are highlighted red or blue and synthetic mismatches in the guide sequence are colored red. (B) Highly specific detection of strain SNPs allows for the differentiation of ZIKV African versus American RNA targets using Cas13a. (n=2 technical replicates, two-tailed Student t-test; **, p < 0.01; ***, p < 0.001; bars represent mean ± s.e.m.) (C) Schematic of DENV strain target regions and the crRNA sequences used for detection. SNPs in the target are highlighted red or blue and synthetic mismatches in the guide sequence are colored red. (D) Highly specific detection of strain SNPs allows for the differentiation of DENV strain 1 versus strain 3 RNA targets using Cas13a. (n=2 technical replicates, two-tailed Student t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; bars represent mean ± s.e.m.)

Figure 4. SHERLOCK can discriminate SNPs for human genotyping and cell-free allele DNA detection

(A) Circos plot showing location of human SNPs detected with SHERLOCK. (B) SHERLOCK can correctly genotype four different individuals at four different SNP sites in the human genome. The genotypes for each individual and identities of allele-sensing crRNAs are annotated below each plot. (n=4 technical replicates, two-tailed Student t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; bars represent mean ± s.e.m.) (C) Schematic of cell-free DNA detection of cancer mutations using SHERLOCK. (D) Sequences of two genomic loci assayed for cancer mutations in cell-free DNA. Shown are the target genomic sequence with the SNP highlighted in blue and the mutant/wild-type sensing crRNA sequences with synthetic mismatches colored in red. (E,F) Cas13a can detect the mutant minor allele in mock cell-free DNA samples for the EGFR L858R (E) or the BRAF V600E (F) minor allele. (n=4 technical replicates, two-tailed Student t-test; *, p < 0.05; **, p < 0.01; ****, p < 0.0001; bars represent mean ± s.e.m.)