Abstract
Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, the MegaTev enzyme generates two double-strand breaks to excise an intervening 30-bp fragment. In HEK 293 cells, we observe a high frequency of gene disruption without co-expression of DNA end-processing enzymes. Deep sequencing of disrupted target sites revealed minimal processing, consistent with the MegaTev sequestering the double-strand breaks from the DNA repair machinery. Off-target profiling revealed no detectable cleavage at sites where the I-TevI CNNNG cleavage motif is not appropriately spaced from the I-OnuI binding site. The MegaTev enzyme represents a small, programmable nuclease platform for extremely specific genome-engineering applications.
Highlights
The rapid pace of development in the genome-editing field has led to a number of competing technologies, each with their benefits and limitations [1,2]
We tested activity on hybrid target sites consisting of the native I-TevI CNNNG cleavage motif (5 -CAACG-3 ) and deoxyribonucleic acid (DNA) spacer derived from the phage T4 thymidylate synthase gene fused to either the I-OnuI E2 or the I-LtrI binding site (TO or TL, respectively) (Figure 1A)
No activity was observed for either fusion on the reciprocal substrates (Figure 1B), showing that the I-TevI nuclease domain does not direct targeting of the MegaTevs
Summary
The rapid pace of development in the genome-editing field has led to a number of competing technologies, each with their benefits and limitations [1,2]. The technologies can be broadly characterized based on the nuclease domain used to introduce a double-strand break (DSB) or nick at a target site. Two common reagents are zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) that utilize the dimeric and non-specific FokI nuclease domain [3,4,5,6]. Two head-to-head ZFN or TALEN pairs must be designed to target a single site and positioned such that the FokI domains can dimerize to introduce a DSB [7,8,9], typically with 4-nt 5 overhangs. The recently developed CRISPR/Cas system has received significant attention due to the ease of programming targeting [13,14]. The I-TevI-based reagents are active on substrates that contain a preferred CNNNG cleavage motif, generating 2-nt 3 overhangs
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