MegaTALs: a novel designer rare-cleaving nuclease scaffold for highly active and specific genome engineering

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Sandrine Boissel, Michael Certo, Barry Stoddard, and Andrew Scharenberg

University of Washington/Seattle Children's Research Institute, United States

Genome engineering applications are currently limited by the specificity and designability of available nuclease platforms. Zinc finger nulceases (ZFNs) and transcription activator-like effector nucleases (TALENs), dimeric proteins formed by fusing the FokI cleavage domain with an auxiliary DNA binding domain, have a greater potential for off-target cleavage due to their distinct DNA binding and cleavage domains and by inappropriate coupling of the nuclease halves. Homing endonucleases (HEs) are monomeric, single domain proteins, making them more reliable reagents for creating single or multiplex DNA cleavage events. Their major limitation, however, is the engineerability of their DNA-binding interface; current design and selection methods often yield HEs with reduced affinity and cleavage activity. We have developed a novel designer rare-cleaving endonuclease platform for genome engineering by fusing DNA-binding transcription activator-like effectors with homing endonucleases. These MegaTALs rescue the activity of low affinity homing endonucleases and improve that of high affinity HEs, boosting repair activity well beyond that of their high affinity HE counterparts. Due to their extended sequence recognition, MegaTALs exhibit higher cleavage activity at their desired target and MegaTALs formed with low affinity HEs demonstrate high target specificity. Furthermore, we found that six TAL effector repeat units were sufficient for achieving maximal activity with a low affinity HE, opening up the possibility for sequence divergence and lentiviral delivery of the MegaTAL gene. We propose that MegaTALs are a novel approach to generating active genome engineering nucleases with high activity and extreme target site specificity.