| Autor: |
Ni, Yuyang, Wang, Yifei, Shi, Xinyu, Yu, Fan, Ruan, Qingmin, Tian, Na, He, Jin, Wang, Xun |
| Zdroj: |
EMBO Reports; Dec2024, Vol. 25 Issue 12, p5316-5330, 15p |
| Abstrakt: |
Retrons, found in bacteria and used for defense against phages, generate a unique molecule known as multicopy single-stranded DNA (msDNA). This msDNA mimics Okazaki fragments during DNA replication, making it a promising tool for targeted gene editing in prokaryotes. However, existing retron systems often exhibit suboptimal editing efficiency. Here, we identify the msd gene in Escherichia coli, which encodes the noncoding RNA template for msDNA synthesis and carries the homologous sequence of the target gene to be edited, as a critical bottleneck. Sequence homology causes the msDNA to bind to the msd gene, thereby reducing its efficiency in editing the target gene. To address this issue, we engineer a retron system that tailors msDNA to the leading strand of the plasmid containing the msd gene. This strategy minimizes msd gene editing and reduces competition with target genes, significantly increasing msDNA availability. Our optimized system achieves very high retron editing efficiency, enhancing performance and expanding the potential for in vivo techniques that rely on homologous DNA synthesis. Synopsis: This study describes the engineering of a retron system in Escherichia coli to optimize multicopy single-stranded DNA (msDNA) for targeted gene editing. The approach enhances editing efficiency by minimizing competition with the msd gene. The msd gene was identified as a critical bottleneck that reduces retron editing efficiency due to sequence homology. Minimizing competition increases msDNA availability, resulting in enhanced gene editing performance. The optimized system achieves the so far highest reported retron editing efficiency. This study describes the engineering of a retron system in Escherichia coli to optimize multicopy single-stranded DNA (msDNA) for targeted gene editing. The approach enhances editing efficiency by minimizing competition with the msd gene. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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