Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.
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 predicti...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4183563?pdf=render |
| _version_ | 1818772882068602880 |
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| author | Regina Cencic Hisashi Miura Abba Malina Francis Robert Sylvain Ethier T Martin Schmeing Josée Dostie Jerry Pelletier |
| author_facet | Regina Cencic Hisashi Miura Abba Malina Francis Robert Sylvain Ethier T Martin Schmeing Josée Dostie Jerry Pelletier |
| author_sort | Regina Cencic |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-18T10:16:24Z |
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| id | doaj.art-def26d93bacc45fb8e941661464e73d9 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-18T10:16:24Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-def26d93bacc45fb8e941661464e73d92022-12-21T21:11:17ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-01910e10921310.1371/journal.pone.0109213Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.Regina CencicHisashi MiuraAbba MalinaFrancis RobertSylvain EthierT Martin SchmeingJosée DostieJerry PelletierThe 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.http://europepmc.org/articles/PMC4183563?pdf=render |
| spellingShingle | Regina Cencic Hisashi Miura Abba Malina Francis Robert Sylvain Ethier T Martin Schmeing Josée Dostie Jerry Pelletier Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. PLoS ONE |
| title | Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. |
| title_full | Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. |
| title_fullStr | Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. |
| title_full_unstemmed | Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. |
| title_short | Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. |
| title_sort | protospacer adjacent motif pam distal sequences engage crispr cas9 dna target cleavage |
| url | http://europepmc.org/articles/PMC4183563?pdf=render |
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