title: MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems.
abstract: Programmable nucleases enable engineering of the genome by utilizing endogenous DNA double-strand break (DSB) repair pathways. Although homologous recombination (HR)-mediated gene knock-in is well established, it cannot necessarily be applied in every cell type and organism because of variable HR frequencies. We recently reported an alternative method of gene knock-in, named the PITCh (Precise Integration into Target Chromosome) system, assisted by microhomology-mediated end-joining (MMEJ). MMEJ harnesses independent machinery from HR, and it requires an extremely short homologous sequence (5-25 bp) for DSB repair, resulting in precise gene knock-in with a more easily constructed donor vector. Here we describe a streamlined protocol for PITCh knock-in, including the design and construction of the PITCh vectors, and their delivery to either human cell lines by transfection or to frog embryos by microinjection. The construction of the PITCh vectors requires only a few days, and the entire process takes ∼ 1.5 months to establish knocked-in cells or ∼ 1 week from injection to early genotyping in frog embryos.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Nakade
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan.
authors.forename: Yuto
authors.initials: Y
authors.lastname: Sakane
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan.
authors.forename: Ken-Ichi T
authors.initials: KT
authors.lastname: Suzuki
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D002875:Chromosomes / Q000235:genetics*; D059766:DNA End-Joining Repair / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D055879:Gene Knock-In Techniques / Q000379:methods*; D006801:Humans; D012689:Sequence Homology, Nucleic Acid
chemical_list: D004262:DNA Restriction Enzymes