title: CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells.
abstract: Gene targeting can be achieved by precise genetic modifications through homology-directed repair (HDR) after DNA breaks introduced by genome editing tools such as CRISPR/Cas9 system. The most common form of HDR is homologous recombination (HR). Binding to the DNA breaks by HR factors is thought to compete with non-homologous end joining (NHEJ), an alternative DNA repair pathway. Here, we knocked out the factors in NHEJ by CRISPR/Cas9 system in silkworm cells, so that increased the activities of HR up to 7-fold. Also efficient HR-mediated genome editing events occurred between the chromosomal BmTUDOR-SN gene and donor DNA sequences with an EGFP gene in the middle of two homologous arms for the target gene. Utilizing the NHEJ-deficient silkworm cells, we found that homologous arms as short as 100 bp in donor DNA could be designed to perform precise genome editing. These studies should greatly accelerate investigations into genome editing of silkworm.
authors.affiliation: Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mon
authors.affiliation: Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.affiliation: Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.
authors.forename: Jae Man
authors.initials: JM
authors.lastname: Lee
authors.affiliation: Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kusakabe
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D012831:Bombyx / Q000166:cytology / Q000235:genetics*; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D059766:DNA End-Joining Repair; D055786:Gene Knockout Techniques / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D049750:Genome, Insect / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015398:Signal Transduction / Q000235:genetics
chemical_list: D019476:Insect Proteins; D049452:Green Fluorescent Proteins