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.
Highlights
Efficient, programmable and site-specific homologous recombination remains a longstanding goal of genetics and genome editing [1]
Our group and others recently demonstrated that the Nterminus of dCas9 could be fused to the FokI nuclease catalytic domain, resulting in a dimeric dCas9-FokI fusion that cleaved DNA sites flanked by two guide RNA-specified sequences [10,11]
We envisioned that the guide RNAs would localize a recCas9 dimer to a gix site flanked by two guide-RNA specified sequences, enabling the Gin domain to catalyze DNA recombination in a guide RNA-programmed manner (Figure 1D)
Summary
Programmable and site-specific homologous recombination remains a longstanding goal of genetics and genome editing [1]. Attempts at directing recombination to loci of interest relied on the transfection of donor DNA with long flanking sequences that are homologous to a target locus [2,3]. This strategy was hampered by very low efficiency and the need for a stringent selection to identify integrants. The efficiency of HDR can be increased if cells are subjected to conditions forcing cell-cycle synchronization or if the enzymes involved in NHEJ are inhibited [15,16,17]. Such conditions can be highly perturbative to cells, limiting potential applications
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