| Autor: |
Kleinstiver, Benjamin P., Prew, Michelle S., Tsai, Shengdar Q., Nguyen, Nhu T., Topkar, Ved V., Zheng, Zongli, Joung, J. Keith |
| Jazyk: |
angličtina |
| Rok vydání: |
2015 |
| Předmět: |
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| Popis: |
CRISPR-Cas9 nucleases are primarily guided by RNA-DNA interactions but also require Cas9-mediated recognition of a protospacer adjacent motif (PAM). While potentially advantageous for specificity, extended PAM sequences limit the targeting range of Cas9 orthologues for genome editing. One possible strategy to relieve this restriction is to relax specificities for certain positions within the PAM. Here we used molecular evolution to modify the NNGRRT PAM specificity of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, shows robust genome editing activities at endogenous human target sites with NNNRRT PAMs. Importantly, using GUIDE-seq, we show that both wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. KKH SaCas9 increased the targeting range of SaCas9 by nearly two- to four-fold. Our molecular evolution strategy does not require structural information and therefore should be applicable to a wide range of Cas9 orthologues. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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