Cas9
清脆的
生物
基因组编辑
基因
遗传学
平菇
突变体
核糖核蛋白
基因组
蘑菇
核糖核酸
食品科学
作者
Tatpong Boontawon,Takehito Nakazawa,Yeon-Jae Choi,Hyeon-Su Ro,Minji Oh,Moriyuki Kawauchi,Masahiro Sakamoto,Yoshio Honda
出处
期刊:Fems Microbiology Letters
[Oxford University Press]
日期:2023-01-01
卷期号:370
被引量:1
标识
DOI:10.1093/femsle/fnad015
摘要
Abstract CRISPR/Cas9 has potential for efficient molecular breeding. Recently, a foreign-DNA-free gene-targeting technology was established by introducing a preassembled Cas9 ribonucleoprotein (RNP) complex into the oyster mushroom Pleurotus ostreatus. However, the target gene was restricted to such a gene like pyrG, since screening of a genome-edited strain was indispensable and could be performed via examination of 5-fluoroorotic acid (5-FOA) resistance caused by the disruption of the target gene. In this study, we simultaneously introduced the Cas9 RNP complex targeting fcy1, a mutation that conferred P. ostreatus resistance to 5-fluorocytosine (5-FC), together with that targeting pyrG. A total of 76 5-FOA resistant strains were isolated during the first screening. Subsequently, a 5-FC resistance examination was conducted, and three strains exhibited resistance. Genomic PCR experiments followed by DNA sequencing revealed that mutations were successfully introduced into fcy1 and pyrG in the three strains. The results indicated that double gene-edited mutants could be obtained in one experiment employing 5-FOA resistance screening for strains with Cas9 RNP incorporation. This work may pave the way for safe CRISPR/Cas9 technology to isolate mutant strains in any gene of interest without an ectopic marker gene.
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