利用CRISPR/Cas9系统检测同源片段长度对重组效率的影响

Evaluation of the relationship between recombination efficiency and the length of homologous fragments using the CRISPR/Cas9 system

目的:在大肠埃希菌中利用CRISPR/Cas9系统对插入失活的m Cherry红色荧光报告基因进行靶向切割并重组,以研究同源片段长度对重组修复效率的影响。方法:通过Golden Gate克隆方法构建Cas9表达质粒p KD46-Cas,转化至大肠埃希菌DH5α中,阿拉伯糖诱导表达Cas9蛋白,Western blot检测蛋白表达。用同样的方法,将一段随机靶标序列引入p Tac-m Cherry-3guid质粒中,造成插入失活,同时在插入序列两侧分别预留0、40、80、120 bp的m Cherry编码区overlap序列,构建出含不同长度同源片段的插入失活m Cherry荧光报告质粒p Q0、p Q40、p Q80、p Q120,并将其分别转化到表达Cas9蛋白的大肠埃希菌DH5α中,对应的细菌平板分别为p Q0组、p Q40组、p Q80组及p Q120组,每组各9个平板,对每个平板的红、白色菌落进行计数,分别计算出红色菌落数/菌落总数的比值,即为重组修复效率。结果:PCR及测序证实p KD46-Cas质粒及插入失活的m Cherry荧光报告载体p Q0、p Q40、p Q80、p Q120构建成功,Western blot鉴定p KD46-Cas在大肠埃希菌DH5α中表达Cas9蛋白;红色菌落计数显示p Q0组为0,其余3组均出现红色菌落,4组重组修复效率的差异有统计学意义(P<0.05),重组修复效率与同源片段长度呈正相关(r=0.792,P<0.01)。结论:成功建立了基于m Cherry红色荧光报告基因和Cas9的同源重组效率检测系统;研究表明不包含同源片段的质粒无法进行有效的重组,当同源片段在0~120 bp时其长度对重组效率有影响。此系统为深入研究原核生物的同源重组提供了一个新的技术平台。

Objective：In this study,CRISPR/Cas9 system was used to target cutting and restructuring of the mCherry red fluorescent reporter gene which inserted an inactivation sequence,and to evaluate the relationship between the length of homologous fragments and recombinant efficiency by the CRISPR/Cas9 system and mCherry reports genes. Methods：Cas9 expression plasmid pKD46-Cas was constructed by Golden Gate clone method and transfected into E.coli DH5α,and the arabinose induced Cas9 protein expression which was detected by Western blot assay. Using Golden Gate clone method, the mCherry red fluorescent reporter gene of pTac- mCherry-3guid plasmid was targeted insertion a random DNA sequence,whose flanking contain 0,40,80 and 120 bp homologues repeats respectivley with mCherry gene to construct the plasmids pQO,pQ40,pQ80,pQ120. This gene insertion caused reporter gene inactivation. The pQ0, pQ40,pQ80 and pQ120 were transformed into E.coli DH5α which expressing Cas9, the corresponding bacteria solid medium were pQ0 group,pQ40 group,pQ80 group and pQ120 group. Then the ratio of the red colonies to the total colonies were statistic to analysis the recombination efficiency. Results：The result of PCR and sequencing analysis showed that pKD46-Cas and the vector pQ0, pQ40,pQ80, pQ120 were correctly constructed. Western blot test demonstrated that Cas9 prote in could successfully express in E.coli DH5α. There was no red colonies in pQ0 group. The difference of recombination efficiency of pQO, pQ40, pQ80, pQ120 group was statistically significant（P〈0.05）,the length of homologous fragments was positive correlated to the recombination efficiency（r=0.792,P〈0.01 ）. Conclusion ：This study developed a new system based on CRISRP/Cas9 and mCherry fluorescent reports gene to detect the efficiency of homologous recombination, recombination may fail without the homologous fragment and the length of the homologous fragment from 0 to 120 bp had effect on the recombination efficiency. This system would be a new technology platform for further study of homologous homologous in prokaryotes.