title: MegaTevs: single-chain dual nucleases for efficient gene disruption.
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.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Wolfs
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
authors.forename: Matthew
authors.initials: M
authors.lastname: DaSilva
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Meister
authors.affiliation: Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5B7, Canada.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5B7, Canada.
authors.forename: Caroline
authors.initials: C
authors.lastname: Schild-Poulter
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada dedgell@uwo.ca.
authors.forename: David R
authors.initials: DR
authors.lastname: Edgell
mesh_terms: D020134:Catalytic Domain; D053903:DNA Breaks, Double-Stranded; D053837:DNA Cleavage; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis; D059372:Nucleotide Motifs; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases; C065074:I-TEVI endonuclease