Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 115, Issue 14, Pages 3669-3673Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1718148115
Keywords
Cas9; tuned gRNA; tgRNA; mutation prevention; synthetic biology
Categories
Funding
- US National Institutes of Health National Human Genome Research Institute Grant [P50 HG005550]
- Wyss Institute for Biologically Inspired Engineering
- National Cancer Institute [5T32CA009216-34]
- Career Award for Medical Scientists from the Burroughs Wellcome Fund
- Defense Threat Reduction Agency [HDTRA1-15-1-0051]
- Paul G. Allen Frontiers Group
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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 ourmethodology 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.
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