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

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Listed:
  • Jeffrey L. Bessen

    (Broad Institute of Harvard and MIT
    Harvard University
    Harvard University)

  • Lena K. Afeyan

    (Broad Institute of Harvard and MIT
    Harvard University
    Harvard University)

  • Vlado Dančík

    (Broad Institute of Harvard and MIT)

  • Luke W. Koblan

    (Broad Institute of Harvard and MIT
    Harvard University
    Harvard University)

  • David B. Thompson

    (Harvard University
    Harvard University)

  • Chas Leichner

    (Google Inc.)

  • Paul A. Clemons

    (Broad Institute of Harvard and MIT)

  • David R. Liu

    (Broad Institute of Harvard and MIT
    Harvard University
    Harvard University)

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.

Suggested Citation

  • Jeffrey L. Bessen & Lena K. Afeyan & Vlado Dančík & Luke W. Koblan & David B. Thompson & Chas Leichner & Paul A. Clemons & David R. Liu, 2019. "High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases," Nature Communications, Nature, vol. 10(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-09987-0
    DOI: 10.1038/s41467-019-09987-0
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