Compact designer TALENs for efficient genomeengineering

摘要

Transcription activator-like effector nucleases are readily targetable ‘molecular scissors’ forgenome engineering applications. These artificial nucleases offer high specificity coupled withsimplicity in design that results from the ability to serially chain transcription activator-likeeffector repeat arrays to target individual DNA bases. However, these benefits come at thecost of an appreciably large multimeric protein complex, in which DNA cleavage is governedby the nonspecific FokI nuclease domain. Here we report a significant improvement to thestandard transcription activator-like effector nuclease architecture by leveraging the partiallyspecific I-TevI catalytic domain to create a new class of monomeric, DNA-cleaving enzymes.In vivo yeast, plant and mammalian cell assays demonstrate that the half-size, single-polypeptidecompact transcription activator-like effector nucleases exhibit overall activity andspecificity comparable to currently available designer nucleases. In addition, we harness thecatalytic mechanism of I-TevI to generate novel compact transcription activator-like effectornuclease-based nicking enzymes that display a greater than 25-fold increase in relativetargeted gene correction efficacy.