Mutants of Cre recombinase with improved accuracy.

Abstract:

:Despite rapid advances in genome engineering technologies, inserting genes into precise locations in the human genome remains an outstanding problem. It has been suggested that site-specific recombinases can be adapted towards use as transgene delivery vectors. The specificity of recombinases can be altered either with directed evolution or via fusions to modular DNA-binding domains. Unfortunately, both wild-type and altered variants often have detectable activities at off-target sites. Here we use bacterial selections to identify mutations in the dimerization surface of Cre recombinase (R32V, R32M and 303GVSdup) that improve the accuracy of recombination. The mutants are functional in bacteria, in human cells and in vitro (except for 303GVSdup, which we did not purify), and have improved selectivity against both model off-target sites and the entire E. coli genome. We propose that destabilizing binding cooperativity may be a general strategy for improving the accuracy of dimeric DNA-binding proteins.

journal_name

Nat Commun

journal_title

Nature communications

authors

Eroshenko N,Church GM

doi

10.1038/ncomms3509

subject

Has Abstract

pub_date

2013-01-01 00:00:00

pages

2509

issn

2041-1723

pii

ncomms3509

journal_volume

4

pub_type

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