Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System

Redding, Sy; Sternberg, Samuel H.; Marshall, Myles; Gibb, Bryan; Bhat, Prashant; Guegler, Chantal K.; Wiedenheft, Blake; Doudna, Jennifer A.; Greene, Eric C.

doi:10.1016/j.cell.2015.10.003

Cited by 185 publications

(296 citation statements)

References 38 publications

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“…3D). This result suggests that target degradation by Cas2/3 does not liberate Csy for another round of target binding, similar to the single-turnover enzyme activity reported for both Cascade (type I-E) and Cas9-mediated (type II-A) DNA cleavage (31,44).…”

Section: Significancesupporting

confidence: 62%

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Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

Rollins

Chowdhury

Carter

et al. 2017

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

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102

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Significance Prokaryotes have adaptive immune systems that rely on CRISPRs (clustered regularly interspaced short palindromic repeats) and diverse CRISPR-associated ( cas ) genes. Cas1 and Cas2 are conserved components of CRISPR systems that are essential for integrating fragments of foreign DNA into CRISPR loci. In type I-F immune systems, the Cas2 adaptation protein is fused to the Cas3 interference protein. Here we show that the Cas2/3 fusion protein from Pseudomonas aeruginosa stably associates with the Cas1 adaptation protein, forming a 375-kDa propeller-shaped Cas1–2/3 complex. We show that Cas1, in addition to being an essential adaptation protein, also functions as a repressor of Cas2/3 nuclease activity and that foreign DNA binding by the CRISPR RNA-guided surveillance complex activates the Cas2/3 nuclease.

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Section: Significancesupporting

confidence: 62%

“…In type I CRISPR systems, the crRNA-guided surveillance complex initiates DNA targeting by binding a short sequence called a Protospacer Adjacent Motif (PAM) (31,32). PAM recognition facilitates crRNA-guided strand invasion, allowing the guide sequence of the crRNA to hybridize to the complementary strand of the foreign DNA.…”

Section: Significancementioning

confidence: 99%

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

Rollins

Chowdhury

Carter

et al. 2017

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

Self Cite

102

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“…Depending on the architecture of the effector-CRISPR RNA (crRNA) interference module, different CRISPR-Cas systems could be assigned into two classes [1]: class 1 systems (multi-subunit complex, such as Cascade) [4,5] and class 2 systems (single enzyme, such as Cas9) [6,7]. Cas9 is the signature member of class 2 systems, which functions as a multi-domain endonuclease, along with crRNA and trans-activating crRNA (tracrRNA), or alternatively with a synthetic single-guide RNA (sgRNA), to cleave both strands of the target DNA [6][7][8].…”

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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“…matching protospacer, which leads to CRISPR interference. Recently, biophysical methods were used to present evidence that effector complexes formed by the E. coli Cascade with partially matching protospacers are structurally different from complexes formed with fully matching protospacers (23) and may stimulate recruitment of adaptation module enzymes (24). Here, we asked whether interactions with fully matching protospacers can support primed adaptation.…”

mentioning

confidence: 99%

Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

Semenova

Savitskaya

Musharova

et al. 2016

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

111

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Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated (Cas) immunity relies on adaptive acquisition of spacers-short fragments of foreign DNA. For the type I-E CRISPR-Cas system from Escherichia coli, efficient "primed" adaptation requires Cas effector proteins and a CRISPR RNA (crRNA) whose spacer partially matches a segment (protospacer) in target DNA. Primed adaptation leads to selective acquisition of additional spacers from DNA molecules recognized by the effector-crRNA complex. When the crRNA spacer fully matches a protospacer, CRISPR interference-that is, target destruction without acquisition of additional spacers-is observed. We show here that when the rate of degradation of DNA with fully and partially matching crRNA targets is made equal, fully matching protospacers stimulate primed adaptation much more efficiently than partially matching ones. The result indicates that different functional outcomes of CRISPR-Cas response to two kinds of protospacers are not caused by different structures formed by the effector-crRNA complex but are due to the more rapid destruction of targets with fully matching protospacers.CRISPR-Cas | CRISPR interference | primed CRISPR adaptation

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Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System

Cited by 185 publications

References 38 publications

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

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

Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

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