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

Abstract:

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 pQ0，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 pQ0，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.