Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA

Yamano, Takashi; Nishimasu, Hiroshi; Zetsche, Bernd; Hirano, Hajime; Slaymaker, Ian M.; Li, Yinqing; Fedorova, Iana; Nakane, Takanori; Makarova, Kira S.; Koonin, Eugene V.; Ishitani, Ryuichiro; Zhang, Feng; Nureki, Osamu

doi:10.1016/j.cell.2016.04.003

Cited by 614 publications

(776 citation statements)

References 41 publications

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“…The structure of the AsCpf1-crRNA-DNA ternary complex, which is similar to a recently reported structure of a closely related ternary complex [25], resembles a bilobal scaffold with an overall "Crab Claw" shape ( Figure 1C and 1D). AsCpf1 can be divided into two lobes: an α-helical recognition (REC) lobe consisting of Helical-I and Helical-II domains, and a NUC lobe consisting of OBD, LHD and RuvC domains, as well as the newly characterized Nuc domain [25] ( Figure 1A, 1C and 1D). The bridge helix motif is inserted between RuvC-I and RuvC-II motifs and connects the REC and NUC lobes from the middle of the whole complex ( Figure 1C).…”

Section: Resultssupporting

confidence: 53%

“…By comparing the recently reported structures of pre-target-bound Cpf1-crR-NA binary complex [23] with target-bound Cpf1-crRNA-DNA ternary complex (this study; see also ref. [25]), we found that Cpf1 employs a unique mechanism for target recognition distinct from that reported for Cas9.…”

Section: Introductionmentioning

confidence: 74%

“…The Cas9-sgRNA pre-target binary conformation was also found to be competent for PAM recognition by forming a preformed PAM-interacting cleft [14]. Whether Cpf1 employs a similar strategy for target recognition is still unknown, although the seed segment of crRNA in Cpf1-crRNA binary complex has been predicted to most likely form the A-form structure [25].…”

Section: Introductionmentioning

confidence: 99%

“…Based on sequence analysis, Cpf1 contains only one detectable RuvC endonuclease domain, which has lead to the initial hypothesis that Cpf1 may form a dimer to cleave the two strands of target DNA [19]. Very recently, structural and functional studies show that Cpf1 acts as a monomer [23][24][25] and contains a second putative novel nuclease (NUC) domain [25]. In addition to the target DNA interference activity, Cpf1 was also found to cleave precursor crRNA (pre-crRNA), leading to the generation of mature crRNAs [24].…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 53%

Section: Introductionmentioning

confidence: 74%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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“…Distant homology searches of the putative Cas proteins were performed using HHpred from the HH-suite 49 . High scoring HHpred hits were used to infer domain architecture based on comparison to solved crystal structures 50,51 , and secondary structure that was predicted by JPred4 52 . Protein modeling was performed using Phyre2 53 .…”

Section: Crispr-cas Computation Analysesmentioning

confidence: 99%

New CRISPR–Cas systems from uncultivated microbes

Burstein

Harrington

Strutt

et al. 2016

Nature

507

369

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CRISPR-Cas systems provide microbes with adaptive immunity by employing short sequences, termed spacers, that guide Cas proteins to cleave foreign DNA 1,2 . Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein bound to RNA recognizes and cleaves targeted sequences 3,4 . The programmable nature of these minimal systems has enabled their repurposing as a versatile technology that is broadly revolutionizing biological and clinical research 5 . However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving untapped the vast majority of enzymes from organisms that have not been cultured. Metagenomics, the sequencing of DNA extracted from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms 6,7 . Here, using genome-resolved metagenomics, we identified novel CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in littlestudied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two

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Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

2018

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Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way.

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Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA

Cited by 614 publications

References 41 publications

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

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

New CRISPR–Cas systems from uncultivated microbes

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

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