High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases.

Jeffrey L Bessen, Lena K Afeyan, Vlado Dančík, Luke W Koblan, David B Thompson, Chas Leichner, Paul A Clemons, David R Liu

Nature communications 2019 Apr 26

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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.

Citation

Jeffrey L Bessen, Lena K Afeyan, Vlado Dančík, Luke W Koblan, David B Thompson, Chas Leichner, Paul A Clemons, David R Liu. High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases. Nature communications. 2019 Apr 26;10(1):1937