title: Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes.
abstract: The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.
authors.affiliation: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors.forename: Bangmei
authors.initials: B
authors.lastname: Wang
authors.affiliation: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors.forename: Kunyu
authors.initials: K
authors.lastname: Li
authors.affiliation: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors.forename: Amy
authors.initials: A
authors.lastname: Wang
authors.affiliation: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors.forename: Michelle
authors.initials: M
authors.lastname: Reiser
authors.affiliation: University of Michigan, Ann Arbor, MI.
authors.forename: Thom
authors.initials: T
authors.lastname: Saunders
authors.affiliation: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.|James A. Haley Veterans' Hospital, Tampa, FL.
authors.forename: Richard F
authors.initials: RF
authors.lastname: Lockey
authors.affiliation: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors.forename: Jia-Wang
authors.initials: JW
authors.lastname: Wang
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D059766:DNA End-Joining Repair / Q000235:genetics*; D055879:Gene Knock-In Techniques; D005818:Genetic Engineering; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism; D017393:RNA Editing / Q000235:genetics; D059767:Recombinational DNA Repair / Q000235:genetics*; D015053:Zygote / Q000378:metabolism
chemical_list: D004247:DNA
keywords: CRISPR cleavage; double-strand break (DSB); embryonic stem (ES) cells; homology-directed-repair (HDR); zygote