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Proc Natl Acad Sci U S A. 2018 Apr 3;115(14):3669-3673. doi: 10.1073/pnas.1718148115. Epub 2018 Mar 19.

Precise Cas9 targeting enables genomic mutation prevention.

Author information

1
Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY 10032; ac4304@cumc.columbia.edu gchurch@genetics.med.harvard.edu.
2
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02115.
3
Department of Genetics, Harvard Medical School, Boston, MA 02115.
4
Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, MA 02139.
5
Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139.
6
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA.
7
Broad Institute of MIT and Harvard, Cambridge, MA 02142.
8
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02115; ac4304@cumc.columbia.edu gchurch@genetics.med.harvard.edu.

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.

KEYWORDS:

Cas9; mutation prevention; synthetic biology; tgRNA; tuned gRNA

PMID:
29555762
PMCID:
PMC5889643
DOI:
10.1073/pnas.1718148115
[Indexed for MEDLINE]
Free PMC Article

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