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 and David R. Liu ()
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Jeffrey L. Bessen: Broad Institute of Harvard and MIT
Lena K. Afeyan: Broad Institute of Harvard and MIT
Vlado Dančík: Broad Institute of Harvard and MIT
Luke W. Koblan: Broad Institute of Harvard and MIT
David B. Thompson: Harvard University
Chas Leichner: Google Inc.
Paul A. Clemons: Broad Institute of Harvard and MIT
David R. Liu: Broad Institute of Harvard and MIT

Nature Communications, 2019, vol. 10, issue 1, 1-13

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-09987-0

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