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CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

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Science  27 Apr 2018:
Vol. 360, Issue 6387, pp. 436-439
DOI: 10.1126/science.aar6245
  • Fig. 1 Cas12a target recognition activates nonspecific ssDNA cleavage.

    (A) Cas12a-crRNA complex binds a dsDNA substrate and generates a 5′-overhang staggered cut by using a single RuvC nuclease. (B and C) Representative M13 ssDNA cleavage time courses with purified LbCas12a (left) and SpCas9 (right) complexed with (B) a guide RNA complementary to M13 phage or (C) a guide RNA and complementary ssDNA activator with no sequence homology to M13 phage. bp, base pair; Eco RI, a restriction endonuclease; dCas12a and dCas9, dead variants; bp, base pair; sgRNA, single-guide RNA.

  • Fig. 2 Kinetics of Cas12a ssDNA trans cleavage.

    (A) Sequence-specific plasmid DNA cleavage reactions by LbCas12a-crRNA complex (top) were introduced to a separate radiolabeled dsDNA or ssDNA substrate of unrelated sequence (bottom); time course represented in minutes. Substrate (S) and nucleotide products (P) were resolved by agarose gel electrophoresis (top) or denaturing polyacrylamide gel electrophoresis (PAGE) (bottom). (B) Target dsDNA or (C) nonspecific ssDNA incubated with molar ratios of LbCas12a-crRNA, as indicated. Each point represents the mean quantified percent cleavage after 30 min at 37°C, at which time the reaction was at completion. Error bars represent mean ± SD, where n = 3 replicates. Yellow coloring indicates PAM. Arrowheads indicate cleavage sites. (D) Representative Michaelis-Menten plot for LbCas12a-catalyzed ssDNA trans cleavage using a dsDNA or ssDNA activator. Measured kcat/Km values reported as mean ± SD, where n = 3 Michaelis-Menten fits. V0, rate of catalysis. Color scheme in (B) and (C) is the same as for (D). In (C), blue indicates nonspecific DNA.

  • Fig. 3 Specificity and conservation of trans-cleavage activation.

    (A) LbCas12a-crRNA in the absence or presence of indicated activator, incubated with a radiolabeled nonspecific ssDNA substrate (S) for 30 min at 37°C; products (P) resolved by denaturing PAGE. (B) Observed trans-cleavage rates for LbCas12a using a ssDNA or dsDNA activator with indicated mismatches; rates represent the average of three different targets measured in triplicate, and error bars represent mean + SD, where n = 9 (three replicates for three independent targets). PT, perfect target; mut PAM, mutated PAM. (C) Radiolabeled cis (complementary) or trans (noncomplementary) substrates were incubated with Cas12a-crRNA or Cas9-sgRNA in the presence or absence of a ssDNA activator for 30 min at 37°C; a cis-dsDNA substrate was used in the “no enzyme” lanes. Substrate (S) and nucleotide products (P) were resolved by denaturing PAGE. NmCas9 is Neisseria meningitidis Cas9.

  • Fig. 4 Rapid identification of HPV16 and HPV18 in human samples by DETECTR.

    (A) Diagram of HPV16 and HPV18 sequences within the hypervariable loop V of the L1-encoding gene targeted by Cas12a; yellow highlighted bases indicate 5′ PAM sequence. (B) Heatmap represents normalized mean fluorescence values of HPV16 and HPV18 detected in human cell lines by DETECTR; normalized scale shown in (D). (C) Schematic outlining DNA extraction from human anal samples to HPV identification by DETECTR. RPA, recombinase polymerase amplification; F, fluorophore; Q, quencher. (D) Identification of HPV16 and HPV18 in 25 patient samples by PCR (left) and DETECTR (right). DETECTR heatmap represents normalized mean fluorescence values.

Supplementary Materials

  • CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

    Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky, Jennifer A. Doudna

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

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    • Materials and Methods
    • Figs. S1 to S15
    • Table S1
    • References

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