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Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro.

ABSTRACT: In type I CRISPR-Cas systems, primed adaptation of new spacers into CRISPR arrays occurs when the effector Cascade-crRNA complex recognizes imperfectly matched targets that are not subject to efficient CRISPR interference. Thus, primed adaptation allows cells to acquire additional protection against mobile genetic elements that managed to escape interference. Biochemical and biophysical studies suggested that Cascade-crRNA complexes formed on fully matching targets (subject to efficient interference) and on partially mismatched targets that promote primed adaption are structurally different. Here, we probed Escherichia coli Cascade-crRNA complexes bound to matched and mismatched DNA targets using a magnetic tweezers assay. Significant differences in complex stabilities were observed consistent with the presence of at least two distinct conformations. Surprisingly, in vivo analysis demonstrated that all mismatched targets stimulated robust primed adaptation irrespective of conformational states observed in vitro. Our results suggest that primed adaptation is a direct consequence of a reduced interference efficiency and/or rate and is not a consequence of distinct effector complex conformations on target DNA.

SUBMITTER: Krivoy A

PROVIDER: S-EPMC5934681 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro.

Krivoy Andrey A Rutkauskas Marius M Kuznedelov Konstantin K Musharova Olga O Rouillon Christophe C Severinov Konstantin K Seidel Ralf R

Nucleic acids research 20180501 8

In type I CRISPR-Cas systems, primed adaptation of new spacers into CRISPR arrays occurs when the effector Cascade-crRNA complex recognizes imperfectly matched targets that are not subject to efficient CRISPR interference. Thus, primed adaptation allows cells to acquire additional protection against mobile genetic elements that managed to escape interference. Biochemical and biophysical studies suggested that Cascade-crRNA complexes formed on fully matching targets (subject to efficient interfer ...[more]

PMID: 29596641

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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

Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro.

Publications

Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro.

Similar Datasets

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