Dataset Information

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

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')NGG(3') protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5' end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.

SUBMITTER: Cencic R

PROVIDER: S-EPMC4183563 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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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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Project description:CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.

Dataset Information

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

Publications

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

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