该研究旨在为解脂耶氏酵母(Yarrowia lipolytica)开发先导编辑(prime editing,PE)系统,优化其功能和编辑效率,为解脂耶氏酵母提供一种新的基因编辑工具。由于解脂耶氏酵母DNA双链断裂(double-strand breaks,DSB)主要是通过非同源末端重组(non-homologous end joining,NHEJ)方式进行修复,导致规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)系统的应用受到限制。而PE系统不依赖供体DNA,不引入双链断裂,以对细胞损伤更小的方式实现基因组DNA的插入、缺失和突变。该研究对CRISPR系统进行改造,首次构建了解脂耶氏酵母先导编辑系统(prime editing in Y.lipolytica,PEyl),并优化先导编辑向导RNA(prime editing guide RNA,pegRNA)的稳定性,能够有效地对基因组进行无痕编辑,实现单碱基精度的删除、插入和置换,为解脂耶氏酵母的遗传改造提供了新的合成生物学工具。此外,研究还应用PEyl系统成功对解脂耶氏酵母的EYD1基因进行移码突变失活,展现了PEyl系统在精准基因编辑中的应用潜力。
唐雅利
,
刘薇
,
姚逸萍
,
栾春光
,
王德良
,
薛洁
,
黄本宸
,
王晖
,
刘树文
,
孟杰
. 基于CRISPR/Cas9的解脂耶氏酵母先导编辑系统PEyl的构建与应用[J]. 食品与发酵工业, 2025
, 51(18)
: 186
-193
.
DOI: 10.13995/j.cnki.11-1802/ts.041513
The research aimed to develop prime editing (PE) system for Yarrowia lipolytica, improved the function and editing efficiency and provided a new genetic editing tool.Given that Y.lipolytica predominantly repairs double-strand breaks (DSBs) through non-homologous end joining (NHEJ), the utility of the clustered regularly interspaced short palindromic repeats (CRISPR) system was limited.In contrast, the PE system did not necessitate a DNA donor and avoided the induction of double-strand breaks, offered a gentler approach for precise insertion, deletion, and mutation of genomic DNA with minimal cellular damage.In this study, we had modified CRISPR system and, for the first time, constructed the PE system in Y.lipolytica (PEyl) successfully, then we optimized the stability of prime editing guide RNA (pegRNA), allowing for effectively editing the genome without collateral damage and enabling DNA deletion, insertion and replacement of single base accuracy.The PE system brought a novel synthetic biology tool for genetic modification of Y.lipolytica.Furthermore, the PEyl system was applied to inactivate the EYD1 gene by frameshift mutation, demonstrating the potential of the PEyl system in precision gene editing in Y.lipolytccica.
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