Publication Date:
2015-06-23
Description:
Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540238/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540238/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kleinstiver, Benjamin P -- Prew, Michelle S -- Tsai, Shengdar Q -- Topkar, Ved V -- Nguyen, Nhu T -- Zheng, Zongli -- Gonzales, Andrew P W -- Li, Zhuyun -- Peterson, Randall T -- Yeh, Jing-Ruey Joanna -- Aryee, Martin J -- Joung, J Keith -- DP1 GM105378/DP/NCCDPHP CDC HHS/ -- DP1 GM105378/GM/NIGMS NIH HHS/ -- R01 GM088040/GM/NIGMS NIH HHS/ -- R01 GM107427/GM/NIGMS NIH HHS/ -- England -- Nature. 2015 Jul 23;523(7561):481-5. doi: 10.1038/nature14592. Epub 2015 Jun 22.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] Molecular Pathology Unit &Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [3] Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA. ; 1] Molecular Pathology Unit &Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. ; 1] Molecular Pathology Unit &Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA [3] Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm SE-171 77, Sweden. ; 1] Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA [3] Broad Institute, Cambridge, Massachusetts 02142, USA. ; Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. ; 1] Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA. ; 1] Molecular Pathology Unit &Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA [2] Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA [3] Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26098369" target="_blank"〉PubMed〈/a〉
Keywords:
Amino Acid Substitution/genetics
;
Animals
;
CRISPR-Associated Proteins/*genetics/*metabolism
;
CRISPR-Cas Systems
;
Cell Line
;
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
;
Directed Molecular Evolution
;
Genome/genetics
;
Humans
;
Mutation/genetics
;
*Nucleotide Motifs
;
Protein Engineering/*methods
;
Staphylococcus aureus/enzymology
;
Streptococcus pyogenes/*enzymology
;
Streptococcus thermophilus/enzymology
;
Substrate Specificity/genetics
;
Zebrafish/embryology/genetics
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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