title: Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.
abstract: The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.
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: Thomas A
authors.initials: TA
authors.lastname: Hamilton
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
authors.forename: Jeremy T
authors.initials: JT
authors.lastname: Lant
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
authors.forename: Marcon
authors.initials: M
authors.lastname: Laforet
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
authors.forename: Jenny
authors.initials: J
authors.lastname: Zhang
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: Louisa M
authors.initials: LM
authors.lastname: Salemi
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Gloor
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: D064113:CRISPR-Cas Systems; D053903:DNA Breaks, Double-Stranded; D053843:DNA Mismatch Repair; D004706:Endodeoxyribonucleases / Q000235:genetics*; D019286:Endopeptidase K / Q000737:chemistry; D004926:Escherichia coli; D017353:Gene Deletion; D000072669:Gene Editing; D016678:Genome; D057809:HEK293 Cells; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D004706:Endodeoxyribonucleases; C065074:I-TEVI endonuclease; D019286:Endopeptidase K
keywords: CRISPR/Cas9; I-TevI homing endonuclease; NHEJ; genome editing