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Nucleic Acids Res DOI:10.1093/nar/gkw707

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells.

Publication TypeJournal Article
Year of Publication2016
AuthorsChaikind, B, Bessen, JL, Thompson, DB, Hu, JH, Liu, DR
JournalNucleic Acids Res
Volume44
Issue20
Pages9758-9770
Date Published2016 Nov 16
ISSN1362-4962
Abstract

We describe the development of 'recCas9', an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases.

DOI10.1093/nar/gkw707
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/27515511?dopt=Abstract

Alternate JournalNucleic Acids Res.
PubMed ID27515511
PubMed Central IDPMC5175349
Grant ListR01 EB022376 / EB / NIBIB NIH HHS / United States
R35 GM118062 / GM / NIGMS NIH HHS / United States