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Sequencing of reaction products from protospacer integration by Cas1-Cas2

ABSTRACT: We report the sites of protospacer integration into plasmids by Cas1-Cas2. The goal of this study is to determine the role of sequence elements on protospacer integration. Methods: Cas1-Cas2 integration products were fragmented, end-repaired, adapter ligated, PCR amplified, and analyzed by Illumina sequencing. Protospacer-containing reads were selected by Cutadapt and mapped to the plasmid with Bowtie.

ORGANISM(S): Cloning vector pUC19

SUBMITTER: James Nunez

PROVIDER: E-GEOD-64552 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

Nuñez James K JK Lee Amy S Y AS Engelman Alan A Doudna Jennifer A JA

Nature 20150218 7542

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substanti ...[more]

PMID: 25707795

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