Abstract
The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.
Highlights
The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities
Site-specific recombinases (SSRs) have the potential to serve as ideal genome editing agents because they directly catalyze the cleavage, strand exchange, and rejoining of DNA fragments at defined recombination targets[1] without relying on the endogenous repair of double-strand breaks, which can induce indels, translocations, other DNA rearrangements, or p53 activation[2,3,4,5]
We sought to develop a system for profiling recombinase specificity through identification of bona fide recombinase substrates from a vast in vitro library of possible targets
Summary
The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. We describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We used Rec-seq specificity profiles to predict offtarget substrates of Tre and Brec[1] recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development. Despite continued efforts to develop SSRs, the challenges of altering their DNA specificity to manipulate arbitrary sequences of interest remains a barrier to their widespread use for genome editing
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