Programmable cas9-recombinase fusion proteins and uses thereof

Abstract

Some aspects of this disclosure provide a fusion protein comprising a guide nucleotide sequence-programmable DNA binding protein domain (e.g., a nuclease-inactive variant of Cas9 such as dCas9), an optional linker, and a recombinase catalytic domain (e.g., a tyrosine recombinase catalytic domain or a serine recombinase catalytic domain such as a Gin recombinase catalytic domain). This fusion protein can recombine DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. The instant disclosure represents a step toward programmable, scarless genome editing inunmodified cells that is independent of endogenous cellular machinery or cell state.

Description

[0001] related application [0002] Pursuant to 35 U.S.C. § 119(e), this application claims priority to U.S. Provisional Patent Applications U.S.S.N. 62 / 372,755 filed on August 9, 2016 and U.S. Provisional Patent Application U.S.S.N. Each of which is incorporated herein by reference. [0003] Government funding [0004] This invention was made with government support under grant numbers R01EB022376 and R35GM118062 awarded by the National Institutes of Health. The government has certain rights in this invention. Background of the invention [0005] Efficient, programmable, and site-specific homologous recombination remains a long-standing goal of genetics and genome editing. Early attempts to direct recombination to a locus of interest relied on transfecting donor DNA with long flanking sequences homologous to the target locus. This strategy is hampered by very low efficiency and thus requires stringent selection to identify integrants. Recent efforts have exploited the ab...

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