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 effici...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.展开更多
拟轮枝镰孢菌(Fusarium verticillioides)是引起玉米茎基腐病和穗粒腐病的主要病原菌之一,严重威胁玉米的产量和品质。为了深入研究拟轮枝镰孢菌致病基因的功能,对该菌中非同源末端连接(non-homologous end joining,NHEJ)途径中的2个关...拟轮枝镰孢菌(Fusarium verticillioides)是引起玉米茎基腐病和穗粒腐病的主要病原菌之一,严重威胁玉米的产量和品质。为了深入研究拟轮枝镰孢菌致病基因的功能,对该菌中非同源末端连接(non-homologous end joining,NHEJ)途径中的2个关键基因FvKu70和FvKu80分别进行了基因敲除以创制高效的基因敲除菌株,并比较了野生型菌株和突变体菌株在营养生长速率、菌落形态、产孢量、对玉米的致病力和基因敲除效率等方面的差异。研究结果表明,FvKu70和FvKu80的基因缺失突变体与野生型FvLNF15-11相比,在PDA平板上的形态特征(如菌丝形态、生长速率、菌落直径、产孢量)没有明显差异,对玉米茎秆的致病力也类似。此外,选择尿嘧啶生物合成相关基因FvpyrG作为敲除的靶基因,分析了FvKu70或FvKu80缺失突变体菌株的同源重组效率,结果显示突变体菌株均显著高于野生型,其中ΔFvKu70的同源重组效率最高。综上所述,FvKu70或FvKu80基因缺失突变体可以快速又高效地实现拟轮枝镰孢菌的基因敲除,为进一步研究该菌的功能基因提供了技术支持。展开更多
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)
文摘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.
文摘在自然界中,植物会遭受各种环境或内源因素导致的DNA损伤,其中DNA双链断裂(double strand breaks,DSBs)的影响最为严重,如果修复不当,将导致基因组不稳定、基因突变甚至细胞死亡。一方面,植物进化出了强大且有序的损伤修复机制,以确保其存活及正常繁衍;另一方面,基于修复过程的容错性及致突变性,T-DNA插入、基因编辑、物理诱变等技术广泛应用于动植物品种改良。相较于哺乳动物,植物DSBs修复通路及其分子机制报道较为有限。本文综述了植物对DSBs损伤的响应、主要修复途径及关键因子,介绍了通路机制尚未完全解析的替代末端连接(alternative end joining,Alt-EJ)的最新研究进展;此外,探讨了重离子束引起的植物DSBs修复特征和多途径选择,以及基于不同DSBs修复途径的基因编辑技术的研究进展,旨在为深入了解植物DSBs损伤响应及修复的分子机制和研发高效生物育种技术提供参考。
文摘拟轮枝镰孢菌(Fusarium verticillioides)是引起玉米茎基腐病和穗粒腐病的主要病原菌之一,严重威胁玉米的产量和品质。为了深入研究拟轮枝镰孢菌致病基因的功能,对该菌中非同源末端连接(non-homologous end joining,NHEJ)途径中的2个关键基因FvKu70和FvKu80分别进行了基因敲除以创制高效的基因敲除菌株,并比较了野生型菌株和突变体菌株在营养生长速率、菌落形态、产孢量、对玉米的致病力和基因敲除效率等方面的差异。研究结果表明,FvKu70和FvKu80的基因缺失突变体与野生型FvLNF15-11相比,在PDA平板上的形态特征(如菌丝形态、生长速率、菌落直径、产孢量)没有明显差异,对玉米茎秆的致病力也类似。此外,选择尿嘧啶生物合成相关基因FvpyrG作为敲除的靶基因,分析了FvKu70或FvKu80缺失突变体菌株的同源重组效率,结果显示突变体菌株均显著高于野生型,其中ΔFvKu70的同源重组效率最高。综上所述,FvKu70或FvKu80基因缺失突变体可以快速又高效地实现拟轮枝镰孢菌的基因敲除,为进一步研究该菌的功能基因提供了技术支持。
