Details:

Alternative Title:
Nature
Personal Author:
Kleinstiver, Benjamin P. ; Prew, Michelle S. ; Tsai, Shengdar Q. ; Topkar, Ved ; Nguyen, Nhu T. ; ... More +
Kleinstiver, Benjamin P. ; Prew, Michelle S. ; Tsai, Shengdar Q. ; Topkar, Ved ; Nguyen, Nhu T. ; Zheng, Zongli ; Gonzales, Andrew P.W. ; Li, Zhuyun ; Peterson, Randall T. ; Yeh, Jing-Ruey Joanna ; Aryee, Martin J. ; Joung, J. Keith ; Less -
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.
Subject:
[+]
Amino Acid Substitution
Animals
Article
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Associated Proteins
CRISPR-Cas Systems
Directed Molecular Evolution
Genome
Humans
Mutation
Nucleotide Motifs
Protein Engineering
Staphylococcus Aureus
Streptococcus Pyogenes
Streptococcus Thermophilus
Substrate Specificity
Zebrafish

Source:
Nature. 523(7561):481-485. ;
Pubmed ID:
26098369
Pubmed Central ID:
PMC4540238
Document Type:
Journal Article ;
Funding:
DP1 GM105378/DP/NCCDPHP CDC HHS/United States ; DP1 GM105378/GM/NIGMS NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R01 GM107427/GM/NIGMS NIH HHS/United States ; ... More +
DP1 GM105378/DP/NCCDPHP CDC HHS/United States ; DP1 GM105378/GM/NIGMS NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R01 GM107427/GM/NIGMS NIH HHS/United States ; R01 GM107427/GM/NIGMS NIH HHS/United States ; Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:18e842357ef5260057fc6b4b13d0d33965c0b8b12b459af5c7741b6f5b3260d1
File Type:

Supporting Files

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