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. 2018 Apr 27;360(6387):436-439.
doi: 10.1126/science.aar6245. Epub 2018 Feb 15.

CRISPR-Cas12a Target Binding Unleashes Indiscriminate Single-Stranded DNase Activity

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Free PMC article

CRISPR-Cas12a Target Binding Unleashes Indiscriminate Single-Stranded DNase Activity

Janice S Chen et al. Science. .
Free PMC article

Abstract

CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.

Figures

Fig. 1.
Fig. 1.. Cas12a target recognition activates non-specific single-stranded DNA cleavage.
(A) Cas12a-crRNA complex binds a dsDNA substrate and generates a 5’ overhang staggered cut using a single RuvC nuclease. (B, C) Representative M13 ssDNA cleavage timecourses with purified LbCas12a (left) and SpCas9 (right) complexed with a (B) guide RNA complementary to M13 phage or (C) a guide RNA and complementary ssDNA activator with no sequence homology to M13 phage.
Fig. 2.
Fig. 2.. Kinetics of Cas12a ssDNA trans-cleavage.
(A) Sequence-specific plasmid DNA cleavage reactions by LbCas12a-crRNA (top) were introduce to a separate radiolabeled dsDNA or ssDNA substrate of unrelated sequence (bottom); timecourses represent minutes. (B) Target dsDNA or (C) non-specific ssDNA incubated with molar ratios of LbCas12a-crRNA as indicated. Each point represents the mean quantified percent cleavage after 30 minutes at 37°C, at which time the reaction was at completion. Error bars represent the mean ± s.d., where n = 3 replicates. (D) Representative Michaelis-Menten plot for LbCas12a-catalyzed ssDNA trans-cleavage using a dsDNA or ssDNA activator. Measured kcat/Km values report mean ± s.d., where n = 3 replicates.
Fig. 3.
Fig. 3.. Specificity and conservation of trans-cleavage activation.
(A) LbCas12a-crRNA in the absence or presence of indicated activator, incubated with a radiolabeled non-specific 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 ± s.d., where n = 9 (three replicates for three independent targets). (C) Radiolabeled cis (complementary) or trans (non-complementary) 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.
Fig. 4.
Fig. 4.. Rapid identification of HPV types 16 and 18 in human samples by DETECTR.
(A) Diagram of HPV16 and HPV18 sequences within the hypervariable loop V of the L1 gene targeted by Cas12a; highlighted bases indicate 5’ PAM sequence. (B) Heatmap represents normalized mean fluorescence values of HPV types 16 and 18 detected in human cell lines by DETECTR; normalized scale represented in (D). (C) Schematic outlining DNA extraction from human anal samples to HPV identification by DETECTR. (D) Identification of HPV types 16 and 18 in 25 patient samples by PCR (left) and DETECTR (right); DETECTR heatmap represents normalized mean fluorescence values.
Fig. 5.
Fig. 5.. Model for PAM-dependent and PAM-independent activation of cis and trans-cleavage by Cas12a.
The Cas12a-crRNA complex binds to a complementary dsDNA in a PAM-dependent manner (top) or ssDNA in a PAM-independent manner (bottom), which is sufficient to unleash indiscriminate ssDNase activity by the RuvC nuclease. Cas12a can also release its PAM-distal cleavage products, which exposes the RuvC active site for multiple rounds of non-specific ssDNA degradation.

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