Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

S, Kim; Loeff, Luuk; Colombo, Sabina; Brouns, Stan J. J.; Joo, Chirlmin

doi:10.1101/608976

Cited by 3 publications

(2 citation statements)

References 70 publications

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“…For example, spacer acquisition in the E. coli Type I-E system requires the integration host factor protein to bend the leader, which sequesters the promoter and most likely shuts off transcription 18 . An integral component of the DNA replisome, DnaJ, is further required to nucleolytically process the PAM-side of the prespacer to complete full-integration 49 . The PSC resolution in this subset of the CRISPR systems may indeed be replication-coupled; however, the coupling does not always take place in a timely fashion, as a small percentage of the E. coli spacers appear to have been acquired from disintegrated prespacer reintegrated in the wrong orientation 37 .…”

Section: Discussionmentioning

confidence: 99%

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

Budhathoki

Xiao

Schuler

et al. 2020

Nat Struct Mol Biol

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Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis ( Efa ) Cas1–Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. Efa Cas1–Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes Efa Cas1–Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex has a surprisingly short mean lifetime. Remarkably, transcription efficiently resolves the postsynaptic complex and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.

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Section: Discussionmentioning

confidence: 99%

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

Budhathoki

Xiao

Schuler

et al. 2020

Nat Struct Mol Biol

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“…2). Moreover, a parallel study on prespacer generation in E. coli also independently demonstrated that the generic nucleases (such as DNA polymerase III or exonuclease T) are sufficient for trimming the prespacers upon PAM recognition by Cas1-2 (74). These explain why the involvement of specific nucleases, such as Cas4, is precluded for prespacer processing in E. coli (see below).…”

Section: Mechanism Of Prespacer Capture and Processing In E Colimentioning

confidence: 95%

Fidelity of prespacer capture and processing is governed by the PAM-mediated interactions of Cas1-2 adaptation complex in CRISPR-Cas type I-E system

Yoganand

Nimkar

et al. 2019

Journal of Biological Chemistry

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Edited by Joel M. Gottesfeld Prokaryotes deploy CRISPR-Cas-based RNA-guided adaptive immunity to fend off mobile genetic elements such as phages and plasmids. During CRISPR adaptation, which is the first stage of CRISPR immunity, the Cas1-2 integrase complex captures invader-derived prespacer DNA and specifically integrates it at the leader-repeat junction as spacers. For this integration, several variants of CRISPR-Cas systems use Cas4 as an indispensable nuclease for selectively processing the protospacer adjacent motif (PAM) containing prespacers to a defined length. Surprisingly, however, a few CRISPR-Cas systems, such as type I-E, are bereft of Cas4. Despite the absence of Cas4, how the prespacers show impeccable conservation for length and PAM selection in type I-E remains intriguing. Here, using in vivo and in vitro integration assays, deep sequencing, and exonuclease footprinting, we show that Cas1-2/I-E-via the type I-Especific extended C-terminal tail of Cas1-displays intrinsic affinity for PAM containing prespacers of variable length in Escherichia coli. Although Cas1-2/I-E does not prune the prespacers, its binding protects the prespacer boundaries from exonuclease action. This ensures the pruning of exposed ends by exonucleases to aptly sized substrates for integration into the CRISPR locus. In summary, our work reveals that in a few CRISPR-Cas variants, such as type I-E, the specificity of PAM selection resides with Cas1-2, whereas the prespacer processing is co-opted by cellular non-Cas exonucleases, thereby offsetting the need for Cas4.Prokaryotes utilize an adaptive immune response mediated by CRISPR and CRISPR-associated proteins (Cas) 2 to respond to infections by mobile genetic elements (MGE) (viz. phages and plasmids) (1-4). CRISPR encompasses a typical architec-This work was supported by Department of Biotechnology (DBT) Grants BT

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Detection of spacer precursors formed in vivo during primed CRISPR adaptation

et al. 2019

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Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.

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Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

Cited by 3 publications

References 70 publications

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

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

Fidelity of prespacer capture and processing is governed by the PAM-mediated interactions of Cas1-2 adaptation complex in CRISPR-Cas type I-E system

Detection of spacer precursors formed in vivo during primed CRISPR adaptation

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