Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

Abstract

Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) that combines isothermal preamplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCK version 2 (SHERLOCKv2) (i) four-channel single-reaction multiplexing with orthogonal CRISPR enzymes; (ii) quantitative measurement of input as low as 2 attomolar; (iii) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxiliary CRISPR-associated enzyme; and (iv) lateral-flow readout. SHERLOCKv2 can detect Dengue or Zika virus single-stranded RNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.

Figure 1. Multiplexed SHERLOCK detection with orthogonal collateral activity of Class 2 enzymes

A) Schematic of assay for determining di-nucleotide preferences of Cas13a/b enzymes. B) Collateral activity of LwaCas13a, CcaCas13b, LbaCas13a, and PsmCas13b on orthogonal di-nucleotide reporters. C) Schematic of collateral activity of Cas12a activated by dsDNA. D) Comparison of collateral activity and pre-amplification enhanced collateral activity (SHERLOCK) of LwaCas13a, PsmCas13b, and AsCas12a. The dotted line denotes 2e9 (aM), the limit of AsCas12a sensitivity without preamplification. Values represent mean +/− S.E.M. E) Schematic of in-sample 4 channel multiplexing using orthogonal Cas13 and Cas12a enzymes. F) In-sample multiplexed detection of ZIKV ssRNA, ssRNA 1, DENV ssRNA, and dsDNA 1 with LwaCas13a, PsmCas13b, CcaCas13b, and AsCas12a. G) Schematic of in-sample multiplexed detection of S. aureus thermonuclease and P. aeruoginosa acyltransferase synthetic targets with LwaCas13a and PsmCas13b. H) In-sample multiplexed RPA and collateral detection at decreasing concentrations of S. aureus thermonuclease and P. aeruoginosa acyltransferase synthetic targets with LwaCas13a and PsmCas13b.

Figure 2. Single molecule quantitation and enhanced signal with SHERLOCK and Csm6

A) Schematic of DNA reaction scheme for quantitation of P. aeroginosa synthetic DNA B) Quantitation of P. aeroginosa synthetic DNA at various RPA primer concentrations. Values represent mean +/− S.E.M. C) Correlation of P. aeroginosa synthetic DNA concentration with detected fluorescence. Values represent mean +/− S.E.M. D) Schematic of independent readout of LwaCas13a and Csm6 cleavage activity with orthogonal reporters. E) Activation of EiCsm6 by LwaCas13a cleavage of adenine-uridine activators with different length adenine tracts. LwaCas13a is targeting synthetic DENV ssRNA. Values represent mean +/− S.E.M. F) Combined LwaCas13a and EiCsm6 signal for increasing concentrations of (A)₆-(U)₅ activator detecting 20nM of DENV ssRNA. Values represent mean +/− S.E.M. G) Kinetics of EiCsm6-enhanced LwaCas13a SHERLOCK detection of P. aeruoginosa acyltransferase synthetic target.

Figure 3. Adapting SHERLOCK for lateral flow detection

A) Schematic of lateral flow detection with SHERLOCK B) Detection of synthetic ZIKV ssRNA using lateral flow SHERLOCK with 1 hour of LwaCas13a reaction C) Quantitation of band intensity from detection in (B) D) Schematic of lateral flow detection of therapeutically relevant EGFR mutations from patient liquid biopsy samples. E) Detection of EGFR L858R mutation in patient-derived cell-free DNA samples with either L858R or WT cancer mutations. Values represent mean +/− S.E.M. F) Lateral-flow detection of EGFR L858R mutation in patient-derived cell-free DNA samples with either L858R or WT alleles. G) Quantitation of band intensity from detection in (E). H) Detection of EGFR exon 19 deletion mutation in patient-derived cell-free DNA samples with either exon 19 deletion or WT alleles. Values represent mean +/− S.E.M. I) Lateral-flow detection of EGFR exon 19 deletion mutation in patient-derived cell-free DNA samples with either exon 19 deletion or WT alleles. J) Quantitation of band intensity from detection in (H). K) Schematic of lateral flow readout of EiCsm6-enhanced LwaCas13a detection of DENV ssRNA L) EiCsm6-enhanced lateral flow detection of synthetic DENV RNA in combination with LwaCas13a without preamplification by RPA. Band intensity quantitation is shown to the right.

Figure 4. Combined therapeutics and diagnostics with Cas13 enzymes

A) Schematic of timeline for detection of disease alleles, correction with REPAIR, and assessment of REPAIR correction. B) Sequences of targets and crRNA designs used for detection of APC alleles. C) Sequences of target and REPAIR guide design used for correction of APC alleles. D) In-sample multiplexed detection of APC alleles from healthy- and disease-simulating samples with LwaCas13a and PsmCas13b. Adjusted crRNA ratio allows for comparisons between different crRNAs that will have different overall signal levels (see supplementary methods for more details). Values represent mean +/− S.E.M. E) Quantitation of REPAIR editing efficiency at the targeted APC mutation. Values represent mean +/− S.E.M. F) In-sample multiplexed detection of APC alleles from REPAIR targeting and non-targeting samples with LwaCas13a and PsmCas13b. Values represent mean +/− S.E.M.