Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

Cencic, Regina; Miura, Hisashi; Malina, Abba; Robert, Françis; Éthier, Sylvain; Schmeing, T.M.; Dostie, Josée; Pelletier, Jerry

doi:10.1371/journal.pone.0109213

Cited by 107 publications

(102 citation statements)

References 33 publications

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“…Our findings provide direct evidence to support previous speculation that Cas9 relies on protospacer sensing to enable accurate targeting 21,22 . In particular, we propose that REC3 binding to the RNA-DNA duplex is necessary for re-orienting REC2, which enables HNH docking at the active site (Extended Data Figure 4h–i).…”

supporting

confidence: 89%

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Chen¹,

Dagdas²,

Kleinstiver³

et al. 2017

Nature

971

877

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The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

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supporting

confidence: 89%

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Chen¹,

Dagdas²,

Kleinstiver³

et al. 2017

Nature

971

877

View full text Add to dashboard Cite

show abstract

“…Although the Cas9-sgRNA complex can bind many off-target sites (56,76,106), with some bearing seed sequences as short as just 5 nt (76,106), only a small fraction of off-target sequences are cleaved (56,106), suggesting that DNA binding per se is insufficient to trigger cleavage of DNA substrates (9). The uncoupling of DNA binding and cleavage indicates that the prokaryotic CRISPR-Cas9 system may use a multistep mechanism for degradation of foreign DNA.…”

Section: Decoupled Dna Binding and Cleavage Eventsmentioning

confidence: 99%

“…The uncoupling of DNA binding and cleavage indicates that the prokaryotic CRISPR-Cas9 system may use a multistep mechanism for degradation of foreign DNA. Indeed, both in vitro and in vivo cleavage assays demonstrate that complementary base pairing at the PAM-distal end is critical for Cas9 cleavage activity (9,24,93). Bulk fluorescence resonance energy transfer (FRET) experiments further suggest that the conformational state of the HNH nuclease domain, which is driven by RNA-DNA hybridization and particularly sensitive to PAM-distal-end complementarity, allosterically controls RuvC activity to ensure accurate, concerted target DNA cleavage (93).…”

Section: Decoupled Dna Binding and Cleavage Eventsmentioning

confidence: 99%

CRISPR–Cas9 Structures and Mechanisms

Jiang

Doudna

2017

Annu. Rev. Biophys.

1,545

1,206

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Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

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“…It has been shown that dCas9 binds to off-target sites although affinity to those sites might be weaker than that to the target sites [22][23][24][25] . There are several ways to manage contamination of molecules bound to those off-target sites.…”

Section: Discussionmentioning

confidence: 99%

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

Fujita

Fujii

2016

JoVE

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The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

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Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

Cited by 107 publications

References 33 publications

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

CRISPR–Cas9 Structures and Mechanisms

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

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