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Precise Cas9 targeting enables genomic mutation prevention

Alejandro Chavez, View ORCID ProfileBenjamin W Pruitt, Marcelle Tuttle, Rebecca S Shapiro, Ryan J Cecchi, Jordan Winston, Brian M Turczyk, Michael Tung, James J Collins, George M Church
doi: https://doi.org/10.1101/058974
Alejandro Chavez
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
2Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
3Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
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Benjamin W Pruitt
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
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  • ORCID record for Benjamin W Pruitt
Marcelle Tuttle
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
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Rebecca S Shapiro
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
4Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
5Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
66, Cambridge, Massachusetts, USA.
7Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
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Ryan J Cecchi
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
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Jordan Winston
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
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Brian M Turczyk
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
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Michael Tung
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
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James J Collins
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
4Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
5Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
66, Cambridge, Massachusetts, USA.
7Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
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  • For correspondence: jimjc@mit.edu gchurch@genetics.med.harvard.edu
George M Church
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
3Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
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  • For correspondence: jimjc@mit.edu gchurch@genetics.med.harvard.edu
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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 a novel 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 designed mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.

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Posted June 14, 2016.
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Precise Cas9 targeting enables genomic mutation prevention
Alejandro Chavez, Benjamin W Pruitt, Marcelle Tuttle, Rebecca S Shapiro, Ryan J Cecchi, Jordan Winston, Brian M Turczyk, Michael Tung, James J Collins, George M Church
bioRxiv 058974; doi: https://doi.org/10.1101/058974
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Precise Cas9 targeting enables genomic mutation prevention
Alejandro Chavez, Benjamin W Pruitt, Marcelle Tuttle, Rebecca S Shapiro, Ryan J Cecchi, Jordan Winston, Brian M Turczyk, Michael Tung, James J Collins, George M Church
bioRxiv 058974; doi: https://doi.org/10.1101/058974

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