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RNA-guided Location and Function of the Cas9 CRISPR Endonuclease in Mammalian Cells

ABSTRACT: The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5’NGG3’ protospacer adjacent motifs (PAM). Examination of dmCas9 binding sites using two Trp53 targeting sgRNAs in Arf -/- MEF cell line (mouse).

ORGANISM(S): Mus musculus

SUBMITTER: Jerry Pelletier

PROVIDER: E-GEOD-57650 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.

Cencic Regina R Miura Hisashi H Malina Abba A Robert Francis F Ethier Sylvain S Schmeing T Martin TM Dostie Josée J Pelletier Jerry J

PloS one 20141002 10

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive ...[more]

PMID: 25275497

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