Real-time observation of CRISPR spacer acquisition by Cas1–Cas2 integrase

Budhathoki, Jagat B.; Xiao, Yibei; Schuler, Gabriel; Hu, Chunyi; Cheng, Alexander H.‐D.; Ding, Fran; Ke, Ailong

doi:10.1038/s41594-020-0415-7

Cited by 22 publications

(28 citation statements)

References 56 publications

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“…Previous studies using in vitro assays from the type II-A system focused on the final step of the integration pathway ( 15 , 23 ) without addressing the mechanisms of integration substrate production. To test whether the Cas9-integrase supercomplex (including Cas1, Cas2, Csn2, Cas9 and guide RNA; hereafter referred to as the supercomplex (SC)) is capable of DNA fragment processing and accurate integration, we devised an acquisition assay in which products formed during in vitro plasmid integration reactions are selected following transformation into E. coli (Figure 2A ).…”

Section: Resultsmentioning

confidence: 99%

OUP accepted manuscript

Jakhanwal

Cress

Maguin

et al. 2021

Nucleic Acids Research

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CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9′s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.

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Section: Resultsmentioning

confidence: 99%

OUP accepted manuscript

Jakhanwal

Cress

Maguin

et al. 2021

Nucleic Acids Research

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“…Many of the major activities associated with CRISPR-Cas mechanisms have been characterised, including nucleolysis and integration, but a number of key enzymes involved in these pathways remain elusive. Although genetic studies have implicated host polymerases in some CRISPR-Cas systems 40 – 42 , CRISPR-specific replicases have not been described so far. Here, we report the characterisation of a family of CRISPR-Cas associated Prim-Pols called CAPPs, which possess a remarkable variety of synthesis activities.…”

Section: Discussionmentioning

confidence: 99%

“…Once CAPP hands over the newly synthesised prespacer to Cas1-Cas2, their association probably persists. We propose that CAPPs may also facilitate the completion of the integration process by disentangling the entwined structure of the fully integrated spacer-repeat intermediate 34 , 42 , 47 using displacement synthesis to both relax and complete replication of this intermediate (Fig. 9d ).…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

et al. 2021

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CRISPR-Cas pathways provide prokaryotes with acquired “immunity” against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.

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“…A recent single molecule FRET analysis suggests that the poststrand transfer complex (or post-synaptic complex; PSC) formed during proto-spacer integration may be resolved by transcription from the CRISPR leader accompanied by limited DNA replication (45). Resolution may also be achieved by replication, although it is argued to be less likely.…”

Section: From Invading Dna To Spacer Deposition At the Crispr Locusmentioning

confidence: 99%

Disintegration promotes proto-spacer integration by the Cas1-Cas2 complex

Javanmardi

Finkelstein

et al. 2020

Preprint

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Abstract‘Disintegration’—the reversal of transposon DNA integration at a target site—is regarded as an abortive off-pathway reaction. Here we challenge this view with a biochemical investigation of the mechanism of protospacer insertion by the Streptococcus pyogenes Cas1-Cas2 complex, which is mechanistically analogous to DNA transposition. In supercoiled target sites, the predominant outcome is the disintegration of one-ended insertions that fail to complete the second integration event. In linear target sites, one-ended insertions far outnumber complete proto-spacer insertions. The second insertion event is most often accompanied by disintegration of the first, mediated either by the 3’-hydroxyl exposed during integration or by water. One-ended integration intermediates may mature into complete spacer insertions via DNA repair pathways that are also involved in transposon mobility. We propose that disintegration-promoted integration is functionally important in the adaptive phase of CRISPR-mediated bacterial immunity, and perhaps in other analogous transposition reactions.

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Real-time observation of CRISPR spacer acquisition by Cas1–Cas2 integrase

Cited by 22 publications

References 56 publications

OUP accepted manuscript

OUP accepted manuscript

CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

Disintegration promotes proto-spacer integration by the Cas1-Cas2 complex

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