Traditionally, knock-in techniques have relied on homologous recombination to drive targeted gene replacement, although other methods using a transposon -mediated system to insert the target gene have been developed. [3] . The use of loxP flanking sites that become excised upon expression of Cre recombinase with gene vectors is an example of this.

Homologous recombination in embryonic stem cells is now a routine method for modifying the mouse genome at a specific locus. The technique was first developed for site-directed mutagenesis in yeast, and has been successfully adapted for mammalian cells (7).

2007年10月25日 · Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the...

Directing CtIP-mediated homologous recombination to the CRISPR/Cas9 cutting site can minimize a global effect following DNA repair. On the other hand, fusions of Cas9 to DN1S (dominant-negative mutant of 53BP1) enhance HDR and precise gene knock-in by inhibiting NHEJ events specifically at Cas9 cleavage sites 86 .

2017年2月20日 · We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97–100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs.

2022年12月22日 · Targeted genome editing events fall into two general classes: knock-in and knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. The majority of cellular repair events will generate these types of mutations, making it an ‘easy’ editing job to get done.

2016年1月20日 · The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show...

2017年5月19日 · Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼ 800 bp of...

2022年4月15日 · a) CRISPR-HDR gene knock-in strategy is based on homologous recombination (HR) between the long homology arms (250–800 bps) in the target genome and the donor vector. Dashed lines refer to the HR event. b) CRIS-PITCh gene KI strategy needs two sgRNA targeting sites on the plasmid donor.

2021年12月9日 · For gene disruption, DNA repair by homologous recombination offers several advantages over nonhomologous end joining, including more efficient screening for knockout clones and improved genomic stability. Here we designed and characterized a knockout fragment intended to repair Cas9-induced gene disruptions by homologous recombination.