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

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.

Modern methods for laboratory diversification of biomolecules

“…These tools have been extended to the realm of directed evolution through engineered deaminase-Cas9 systems to evolve known variants of GFP [76], identify novel mutants conferring resistance to bortezomib [76] and imatinib [77], and to enable robust RNA-programmed recombination through a recombinase-dCas9 fusion [78]. …”

RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

“…A catalytically inactive variant of Cas9 called dCas9 ('dead Cas9', containing the mutations D10A and H840A), has previously been used to target enzymes including transcriptional activators (45)(46)(47), repressors (48,49), base editors (50,51) and others (52,53) to specific target sequences. Using dCas9 as a targeting domain for a transposon could combine this great flexibility with the advantages of integrating vectors.…”

Development of Toolboxes for Precision Genome/Epigenome Editing and Imaging of Epigenetics

“…Further understanding of epigenetic mechanisms is required for specific epigenome regulation; the study of relationships between modification of histone proteins and DNA modification . The concept of hybrid enzymes combining catalytic and DNA binding domains depending on purposes and targets is also important because it enables expansion of scope and variety by minor customization such as use of alternate DNA binding domains in design of recombinases or others ,. Off‐target genome modification is one of the major issues in editing technologies to avoid unexpected side‐effects raised by changing unintended similar sequences.…”