RRC ID 53917
Author Nakade S, Mochida K, Kunii A, Nakamae K, Aida T, Tanaka K, Sakamoto N, Sakuma T, Yamamoto T.
Title Biased genome editing using the local accumulation of DSB repair molecules system.
Journal Nat Commun
Abstract Selective genome editing such as gene knock-in has recently been achieved by administration of chemical enhancer or inhibitor of particular DNA double-strand break (DSB) repair pathways, as well as overexpression of pathway-specific genes. In this study, we attempt to enhance the efficiency further to secure robust gene knock-ins, by using the local accumulation of DSB repair molecules (LoAD) system. We identify CtIP as a strong enhancer of microhomology-mediated end-joining (MMEJ) repair by genetic screening, and show the knock-in-enhancing effect of CtIP LoADing. Next-generation sequencing reveals that CtIP LoADing highly increases the frequency of MMEJ-mediated integration. Selection-free, simultaneous triple gene knock-ins are also achieved with the CtIP-LoADing strategy. Moreover, by replacing the LoADing molecules and targeting strategies, this system can be applied for other specific genome engineering purposes, such as introducing longer deletions for gene disruption, independently introducing multiple mutations without chromosomal deletion, and efficiently incorporating a single-stranded oligodeoxynucleotide donor.
Volume 9(1)
Pages 3270
Published 2018-8-16
DOI 10.1038/s41467-018-05773-6
PII 10.1038/s41467-018-05773-6
PMID 30115916
PMC PMC6095859
MeSH Base Sequence Chromosomes, Human / genetics DNA Breaks, Double-Stranded* DNA End-Joining Repair / genetics DNA Repair / genetics* Gene Editing* Gene Knock-In Techniques HEK293 Cells HeLa Cells Humans
IF 12.353
Resource
Human and Animal Cells CHO-K1(RCB0285)