Structures and mechanisms of CRISPR RNA-guided effector nucleases

Nishimasu, Hiroshi; Nureki, Osamu

doi:10.1016/j.sbi.2016.11.013

Cited by 75 publications

(47 citation statements)

References 65 publications

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“…While dsDNA target recognition has been extensively analyzed in the Cse (Type I-E) system via structural studies (Hayes et al, 2016; Hochstrasser et al, 2014; Jackson et al, 2014; Mulepati et al, 2014; van Erp et al, 2015; Wiedenheft et al, 2011; Zhao et al, 2014), there is enormous variation among CRISPR systems in both sequence and structure and the degree to which these mechanisms and structural motifs are conserved or plastic are only beginning to be explored (Cass et al, 2015; Jackson and Wiedenheft, 2015; Nishimasu and Nureki, 2017; van der Oost et al, 2014). Here, we report cryo-EM structures for the P. aeruginosa Csy complex before and after binding to either dsDNA or the phage-derived inhibitors AcrF1, AcrF2 or AcrF10.…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

Guo

Bartesaghi

et al. 2017

Cell

157

220

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Summary Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryo-EM, we show that the binding of target double-stranded DNA (dsDNA) to a Type I-F Csy surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally-encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveal mechanistic details underlying target recognition and inhibition.

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Section: Discussionmentioning

confidence: 99%

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

Guo

Bartesaghi

et al. 2017

Cell

157

220

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“…In CRISPR-Cas systems, this involves a complicated set of conformational changes in both the DNA substrate and the surveillance complex (Jiang and Doudna, 2015; Nishimasu and Nureki, 2016). The available structure snapshots only captured the apo and the post-R-loop states, but did not resolve the sequence of events in between.…”

Section: Introductionmentioning

confidence: 99%

Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System

Xiao

Luo

Hayes

et al. 2017

Cell

162

261

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Summary Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca Type I-E Cascade: 1) unwinding 11-bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and 2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in Type I CRISPR-Cas systems.

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“…Most of the CRISPR-Cas effector nucleases require a specific sequence next to the target site, called the protospacer adjacent motif (PAM), to initiate the target recognition (Garneau et al, 2010). The CRISPR-Cas system is divided into two classes, class 1 and class 2 (Makarova et al, 2015; Nishimasu and Nureki, 2017; Shmakov et al, 2017). The class 1 effectors comprise multiple Cas proteins, whereas the class 2 effectors contain a single Cas protein.…”

Section: Introductionmentioning

confidence: 99%

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

et al. 2017

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Summary The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA), and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for the target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA, containing either TTTA, TCTA, TCCA or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent crRNA-guided DNA cleavage by the Cpf1 family nucleases.

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Structures and mechanisms of CRISPR RNA-guided effector nucleases

Cited by 75 publications

References 65 publications

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

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