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Precise Cas9 targeting enables genomic mutation prevention.


ABSTRACT: Here, we present a generalized method of guide RNA "tuning" that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.

SUBMITTER: Chavez A 

PROVIDER: S-EPMC5889643 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Precise Cas9 targeting enables genomic mutation prevention.

Chavez Alejandro A   Pruitt Benjamin W BW   Tuttle Marcelle M   Shapiro Rebecca S RS   Cecchi Ryan J RJ   Winston Jordan J   Turczyk Brian M BM   Tung Michael M   Collins James J JJ   Church George M GM   Church George M GM  

Proceedings of the National Academy of Sciences of the United States of America 20180319 14


Here, we present a generalized method of guide RNA "tuning" that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention con  ...[more]

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