Rec-seq: High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases

Rec-seq identifies bona fide recombinase substrates from a pool of randomized DNA sequences through in vitro selection and high-throughput sequencing. We used Rec-seq to study the DNA recombination profiles of several natural and evolved SSRs including Cre, evolved variants of Cre (Tre and Brec1), and other SSR family members (Dre, VCre, and Bxb1). Rec-seq profiling of these enzymes and targeted mutants thereof provide evidence for previously uncharacterized SSR interactions and inform a model for productive recombination in which sufficient protein:DNA interactions are required for catalysis.