Abstract
A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination activity in marmoset ESCs. Here, we compared gene expression in marmoset and human pluripotent stem cells using transcriptomic and quantitative PCR analyses and found that five HR-related genes (BRCA1, BRCA2, RAD51C, RAD51D, and RAD51) were upregulated in marmoset cells. A total of four of these upregulated genes enhanced HR efficiency with CRISPR-Cas9 in human pluripotent stem cells. Thus, the present study provides a novel insight into species-specific mechanisms for the choice of DNA repair pathways.
Highlights
Repairing DNA double strand breaks (DSBs) is indispensable for maintaining genomic stability [1]
In Human pluripotent stem cells (hPSCs), several studies have shown that the homologous recombination (HR) ratio is less than 50%, generally around 30%, even with use of site-specific nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9 [31–34]
Through comparative analyses of gene expression in human and marmoset PSCs, we have identified four genes (RAD51C, RAD51D, BRCA1, and BRCA2) whose single and multiple overexpression increased HR ratios in hiPSCs
Summary
Repairing DNA double strand breaks (DSBs) is indispensable for maintaining genomic stability [1]. The choice of which repair pathways are employed relies on a variety of factors, including DSB complexity, cell type, cell cycle, and species [1–4]. The genome editing CRISPR-Cas technology [5] relies on endogenous pathways for repairing DSBs in cells [6]. The combination of CRISPR-Cas and double-stranded DNA (dsDNA) donor enables precisely targeted integration or deletion of long sequences by HDR, the efficiency of this process is limited by competing DSB repair pathways, mainly NHEJ. Modification of Cas by fusion with DSB repair-related factors such as RAD52, CtIP, MRE11A [11–13], or a dominant negative mutant of P53BP1 [14] can improve HDR efficiency. Overexpression of several DSB-related genes, including RAD52 [12] and RAD18 [15], reportedly contributed to enhancement of Cas9-mediated HDR efficiency
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