Report

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

See allHide authors and affiliations

Science  27 Apr 2018:
Vol. 360, Issue 6387, pp. 439-444
DOI: 10.1126/science.aaq0179
  • Fig. 1 Multiplexed SHERLOCK detection with orthogonal collateral activity of class 2 enzymes.

    (A) Schematic of assay for determining dinucleotide preferences of Cas13a/b enzymes. (B) Collateral activity of LwaCas13a, CcaCas13b, LbaCas13a, and PsmCas13b on orthogonal dinucleotide reporters. (C) Schematic of collateral activity of Cas12a activated by double-stranded DNA (dsDNA). (D) Comparison of collateral activity and preamplification 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 ± SEM. (E) Schematic of in-sample four-channel multiplexing with 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, respectively. (G) Schematic of in-sample multiplexed detection of S. aureus thermonuclease and P. aeruginosa acyltransferase synthetic targets with LwaCas13a and PsmCas13b. (H) In-sample multiplexed RPA and collateral detection at decreasing concentrations of S. aureus thermonuclease and P. aeruginosa acyltransferase synthetic targets with LwaCas13a and PsmCas13b.

  • Fig. 2 Single-molecule quantitation and enhanced signal with SHERLOCK and Csm6.

    (A) Schematic of DNA reaction scheme for quantitation of P. aeruginosa synthetic DNA. (B) Quantitation of P. aeruginosa synthetic DNA at various RPA primer concentrations. Values represent mean ± SEM. (C) Correlation of P. aeruginosa synthetic DNA concentration with detected fluorescence. Values represent mean ± SEM. (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 adenine tracts of different lengths. LwaCas13a is targeting synthetic DENV ssRNA. Values represent mean ± SEM. (F) Combined LwaCas13a and EiCsm6 signal for increasing concentrations of (A)6-(U)5 activator detecting 20 nM of DENV ssRNA. Values represent mean ± SEM. (G) Kinetics of EiCsm6-enhanced LwaCas13a SHERLOCK detection of P. aeruginosa acyltransferase synthetic target.

  • Fig. 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 cfDNA samples with either L858R or wild-type (WT) alleles. Values represent mean ± SEM. (F) Lateral-flow detection of EGFR L858R mutation in patient-derived cfDNA 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 cfDNA samples with either exon 19 deletion or WT alleles. Values represent mean ± SEM. (I) Lateral-flow detection of EGFR exon 19 deletion mutation in patient-derived cfDNA 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.

  • Fig. 4 Combined therapeutics and diagnostics with Cas13 enzymes.

    (A) Schematic of time line 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 ± SEM. (E) Quantitation of REPAIR editing efficiency at the targeted APC mutation. Values represent mean ± SEM. (F) In-sample multiplexed detection of APC alleles from REPAIR targeting and nontargeting samples with LwaCas13a and PsmCas13b. Values represent mean ± SEM.

Supplementary Materials

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

    Jonathan S. Gootenberg, Omar O. Abudayyeh, Max J. Kellner, Julia Joung, James J. Collins, Feng Zhang

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S36
    • Tables S1 to S9
    • References

Navigate This Article