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Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system.

ABSTRACT: CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign nucleic acids. In Escherichia coli, immunity is acquired upon integration of 33-bp spacers into CRISPR arrays. DNA targets complementary to spacers get degraded and serve as a source of new spacers during a process called primed adaptation. Precursors of such spacers, prespacers, are ~33-bp double-stranded DNA fragments with a ~4-nt 3' overhang. The mechanism of prespacer generation is not clear. Here, we use FragSeq and biochemical approaches to determine enzymes involved in generation of defined prespacer ends. We demonstrate that RecJ is the main exonuclease trimming 5' ends of prespacer precursors, although its activity can be partially substituted by ExoVII. The RecBCD complex allows single strand-specific RecJ to process double-stranded regions flanking prespacers. Our results reveal intricate functional interactions of genome maintenance proteins with CRISPR interference and adaptation machineries during generation of prespacers capable of integration into CRISPR arrays.

SUBMITTER: Shiriaeva AA

PROVIDER: S-EPMC9699676 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Host nucleases generate prespacers for primed adaptation in the <i>E. coli</i> type I-E CRISPR-Cas system.

Shiriaeva Anna A AA Kuznedelov Konstantin K Fedorov Ivan I Musharova Olga O Khvostikov Timofey T Tsoy Yuliya Y Kurilovich Elena E Smith Gerald R GR Semenova Ekaterina E Severinov Konstantin K

Science advances 20221125 47

CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign nucleic acids. In <i>Escherichia coli</i>, immunity is acquired upon integration of 33-bp spacers into CRISPR arrays. DNA targets complementary to spacers get degraded and serve as a source of new spacers during a process called primed adaptation. Precursors of such spacers, prespacers, are ~33-bp double-stranded DNA fragments with a ~4-nt 3' overhang. The mechanism of prespacer generation is not clear. Here, we use Fr ...[more]

PMID: 36427302

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Project description:In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5' end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing.IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. In contrast, we show that targeted degradation and acquisition of new immunity elements require highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets.

Dataset Information

Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system.

Publications

Host nucleases generate prespacers for primed adaptation in the <i>E. coli</i> type I-E CRISPR-Cas system.

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Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system.

Publications

Host nucleases generate prespacers for primed adaptation in the &lt;i&gt;E. coli&lt;/i&gt; type I-E CRISPR-Cas system.

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OmicsDI is part of the ELIXIR infrastructure

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Host nucleases generate prespacers for primed adaptation in the <i>E. coli</i> type I-E CRISPR-Cas system.