Crystal Structure of Staphylococcus aureus Cas9

Nishimasu, Hiroshi; Cong, Le; Yan, Wang‐Ji; Ran, F. Ann; Zetsche, Bernd; Li, Yinqing; Kurabayashi, Arisa; Ishitani, Ryuichiro; Zhang, Feng; Nureki, Osamu

doi:10.1016/j.cell.2015.08.007

Cited by 379 publications

(475 citation statements)

References 41 publications

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“…In contrast, the Cas9 phosphate lock loop interaction with the target strand +1 phosphate is linked to target DNA/sgRNA pairing immediately upstream of PAM but does not play a significant role in the initial duplex destabilization. Thus, the Cas9/sgRNA interactions with PAM-proximal sequences of DNA probes not complementary to sgRNA studied here differ from those interactions revealed in crystal complexes, where the target DNA strand was hybridized with the complementary sgRNA (16,(26)(27)(28). Overlays of Cas9/sgRNA-DNA and Cas9/sgRNA structures reveal that in the Cas9/sgRNA structure, domains 2 and 3 of the helical recognition lobe sterically occlude the central channel in which the RNA/DNA hybrid is located in the Cas9/sgRNA-DNA complexes (16,21,26).…”

Section: Discussionmentioning

confidence: 54%

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Mekler

Minakhin

Severinov

2017

Proc. Natl. Acad. Sci. U.S.A.

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The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves doublestranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/ single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/ guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.Cas9 | CRISPR | DNA interrogation | protein-DNA interactions | fluorescence spectroscopy P rokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) gene systems acquire fragments of foreign DNA (protospacers) and insert them as spacers into the CRISPR array in the prokaryotic host genome. Upon subsequent encounters, the complex of Cas proteins with CRISPR RNA (crRNA) bearing the spacer sequence binds and cleaves foreign DNA containing the matching sequence (1-3), thus providing the host organism with adaptive hereditable immunity. The DNA endonuclease Cas9 of type II CRISPR-Cas systems is targeted to specific DNA sequences by a 20-base crRNA spacer that binds to the complementary strand of protospacer DNA, displacing the noncomplementary strand to form an R-loop (4, 5). Both binding and cleavage of target DNA by the Cas9/crRNA complex require a short protospacer adjacent motif (PAM) located immediately downstream of the targeted sequence (6). For the most commonly used Streptococcus pyogenes Cas9 (SpCas9), the PAM sequence is 5′-NGG (7). The perfect match between guide RNA and seven to 12 bases of target DNA at the immediate 5′ side of PAM ("seed" region) is the most critical for DNA binding and cleavage, although limited mispairing in the distal bases is tolerated (8). Two RNA molecules (crRNA and a transactivating crRNA (tracrRNA)) required to guide SpCas9 to targets can be replaced with...

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Section: Discussionmentioning

confidence: 54%

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Mekler

Minakhin

Severinov

2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Structural comparison of available structures of Cas9 orthologs, including the apo structure of Actinomyces naeslundii Cas9 (AnaCas9) from subtype II-C (50) and the DNA-bound structures of SaCas9 (73) and FnCas9 (34), reveals a relatively conserved catalytic core and a highly conserved characteristic arginine-rich bridge helix, as well as the least conserved alpha-helical REC lobe essential for guide RNA binding and a divergent CTD that is responsible for both the PAM recognition and the guide RNA repeat-antirepeat heteroduplex binding (Figure 7b-f ), which explains their distinct PAM specificities and orthologous sgRNA recognition and further enhances our understanding of the structural conservation and divergence among the CRISPR-Cas9 systems. Intriguingly, similar to SpyCas9, both SaCas9 and FnCas9 employ the phosphate lock loop to stabilize the +1 phosphate in the target DNA strand and specifically recognize the major groove of the PAM-proximal duplex via arginine-mediated basespecific interactions (Figure 7d, f ), suggesting the conserved RNA-guided DNA targeting and cleavage mechanisms across the CRISPR-Cas9 systems (34,73).…”

Section: Figurementioning

confidence: 99%

CRISPR–Cas9 Structures and Mechanisms

Jiang

Doudna

2017

Annu. Rev. Biophys.

1,480

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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“…CRISPR-Cas9 has been extensively used for genome editing in various cell types and organisms [11,12]. A series of structural studies of Streptococcus pyogenes Cas9 (SpyCas9) and its orthologs have revealed the detailed intermolecular interactions, as well as the conformational changes among different substrate-bound states [13][14][15][16][17][18].…”

Section: Introductionmentioning

confidence: 99%

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

et al. 2016

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CRISPR-Cas9 and CRISPR-Cpf1 systems have been successfully harnessed for genome editing. In the CRIS-PR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpf1 system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with crRNA and target DNA. Structural comparison between the AsCpf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf1)-crRNA binary complex identifies a unique mechanism employed by Cpf1 for target recognition. The seed sequence required for initial DNA interrogation is disordered in the Cpf1-cRNA binary complex, but becomes ordered upon ternary complex formation. Further, the PAM interacting cleft of Cpf1 undergoes an "open-to-closed" conformational change upon target DNA binding, which in turn induces structural changes within Cpf1 to accommodate the ordered A-form seed RNA segment. This unique mechanism of target recognition by Cpf1 is distinct from that reported previously for Cas9.

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Crystal Structure of Staphylococcus aureus Cas9

Cited by 379 publications

References 41 publications

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

CRISPR–Cas9 Structures and Mechanisms

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

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