Increasing CRISPR/Cas9-mediated gene editing efficiency in T7 phage by reducing the escape rate based on insight into the survival mechanism  

作　　者：Mingjun Sun Jie Gao Hongjie Tang Ting Wu Qinqin Ma Suyi Zhang Yong Zuo Qi Li 

机构地区：[1]College of Life Sciences,Sichuan Normal University,Chengdu 610101,China [2]Luzhou Laojiao Co,Ltd,Luzhou 646000,China [3]National Engineering Research Center of Solid-State Brewing,Luzhou 646000,China

出　　处：《Acta Biochimica et Biophysica Sinica》2024年第6期937-944,共8页生物化学与生物物理学报（英文版）

基　　金：supported by the grants from Luzhou Laojiao Co.,Ltd.(No.2022HX07);Sichuan Science and Technology Program(Nos.2021YJ0273,and 24NSFSC1247);Key Laboratory of Synthetic Biology,Chinese Academy of Sciences.

摘　　要：Bacteriophages have been used across various fields,and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages.However,some bacteriophages can escape from the cleavage of Cas protein,such as Cas9,and decrease the efficiency of genome editing.This study focuses on the bacteriophage T7,which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated.First,we test the escape rates of T7 phage at different cleavage sites,ranging from 10-2 to 10-5.The sequencing results show that DNA point mutations and microhomology-mediated end joining(MMEJ)at the target sites are the main causes.Next,we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region.Moreover,we also knock out the ATP-dependent DNA ligase 1.3 gene,which may be involved in the MMEJ event,and the frequency of MMEJ at 4.3 is reduced from 83%to 18%.Finally,the genome editing efficiency in T7Δ1.3 increases from 20%to 100%.This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1.3 can reduce MMEJ events and enhance gene editing efficiency.These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.

关 键 词：CRISPR Cas9 T7 phage gene editing ESCAPE 

分 类 号：Q78[生物学—分子生物学]