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Nucleic acid detection with CRISPR-Cas13a/C2c2

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Science  28 Apr 2017:
Vol. 356, Issue 6336, pp. 438-442
DOI: 10.1126/science.aam9321
  • Fig. 1 SHERLOCK is capable of single-molecule nucleic acid detection.

    (A) Schematic of SHERLOCK. dsDNA, double-stranded DNA; RT-RPA, reverse transcriptase–RPA. (B) Schematic of ssRNA target detected with 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 ± SEM. (D) SHERLOCK is also capable of single-molecule DNA detection. n = 4 technical replicates; bars represent mean ± SEM.

  • Fig. 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. (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. (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. (G) Schematic of how SHERLOCK is used to distinguish bacterial strains with a universal 16S rRNA gene V3 RPA primer set. (H) SHERLOCK achieves sensitive and specific detection of E. coli or P. aeruginosa gDNA. Ec, E. coli; Kp, K. pneumoniae; Pa, P. aeruginosa; Mt, Mycobacterium tuberculosis; Sa, Staphylococcus aureus. (B, D, F, and H) n = 4 technical replicates, two-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; n.d., not detected; bars represent mean ± SEM.

  • Fig. 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 in red. (B) Highly specific detection of strain SNPs allows for the differentiation of ZIKV African versus American RNA targets with Cas13a. (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 in red. (D) Highly specific detection of strain SNPs allows for the differentiation of DENV strain 1 versus strain 3 RNA targets with Cas13a. (B and D) n = 2 technical replicates, two-tailed Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001; bars represent mean ± SEM.

  • Fig. 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. (C) Schematic of cfDNA detection of cancer mutations using SHERLOCK. (D) Sequences of two genomic loci assayed for cancer mutations in cfDNA. Shown are the target genomic sequences with the SNPs highlighted in blue and the mutant- and wild type–sensing crRNA sequences with synthetic mismatches in red. (E and F) Cas13a can detect the mutant minor allele in mock cfDNA samples for the EGFR L858R (E) or the BRAF V600E (F) minor allele. (B, E, and F) n = 4 technical replicates, two-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; bars represent mean ± SEM.

Supplementary Materials

  • Nucleic acid detection with CRISPR-Cas13a/C2c2

    Jonathan S. Gootenberg, Omar O. Abudayyeh, Jeong Wook Lee, Patrick Essletzbichler, Aaron J. Dy, Julia Joung, Vanessa Verdine, Nina Donghia, Nichole M. Daringer, Catherine A. Freije, Cameron Myhrvold, Roby P. Bhattacharyya, Jonathan Livny, Aviv Regev, Eugene V. Koonin, Deborah T. Hung, Pardis C. Sabeti, James J. Collins, Feng Zhang

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

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    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S13
    • Tables S1 to S7
    • References

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