摘要
Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference(CRISPRi) and Natronobacterium gregoryi Argonaute(NgAgo) interference(NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase(PNKP), DNA ligase IV(LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted(P<0.05) the efficiency of HR to 1.85-and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase(P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed(P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve(P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9(dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.
Non-homologous end-joining(NHEJ) is a predominant pathway for the repair of DNA double-strand breaks(DSB). It inhibits the efficiency of homologous recombination(HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference(CRISPRi) and Natronobacterium gregoryi Argonaute(NgAgo) interference(NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase(PNKP), DNA ligase IV(LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted(P<0.05) the efficiency of HR to 1.85-and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase(P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed(P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve(P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9(dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.
基金
supported by the National Science and Technology Major Project for Breeding of New Transgenic Organisms, China (2016ZX08006002)
the Guangdong Province "Flying Sail Program" Postdoctoral Foundation, China (2016)
