{"title":"预组装Cas9核糖核蛋白双基因靶向在食用菌平菇中的安全基因组编辑","authors":"Tatpong Boontawon, Takehito Nakazawa, Yeon-Jae Choi, Hyeon-Su Ro, Minji Oh, Moriyuki Kawauchi, Masahiro Sakamoto, Yoichi Honda","doi":"10.1093/femsle/fnad015","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":null,"pages":null},"PeriodicalIF":2.2000,"publicationDate":"2023-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroom Pleurotus ostreatus.\",\"authors\":\"Tatpong Boontawon, Takehito Nakazawa, Yeon-Jae Choi, Hyeon-Su Ro, Minji Oh, Moriyuki Kawauchi, Masahiro Sakamoto, Yoichi Honda\",\"doi\":\"10.1093/femsle/fnad015\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":12214,\"journal\":{\"name\":\"Fems Microbiology Letters\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.2000,\"publicationDate\":\"2023-01-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Fems Microbiology Letters\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1093/femsle/fnad015\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Fems Microbiology Letters","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1093/femsle/fnad015","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroom Pleurotus ostreatus.
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.
期刊介绍:
FEMS Microbiology Letters gives priority to concise papers that merit rapid publication by virtue of their originality, general interest and contribution to new developments in microbiology. All aspects of microbiology, including virology, are covered.
2019 Impact Factor: 1.987, Journal Citation Reports (Source Clarivate, 2020)
Ranking: 98/135 (Microbiology)
The journal is divided into eight Sections:
Physiology and Biochemistry (including genetics, molecular biology and ‘omic’ studies)
Food Microbiology (from food production and biotechnology to spoilage and food borne pathogens)
Biotechnology and Synthetic Biology
Pathogens and Pathogenicity (including medical, veterinary, plant and insect pathogens – particularly those relating to food security – with the exception of viruses)
Environmental Microbiology (including ecophysiology, ecogenomics and meta-omic studies)
Virology (viruses infecting any organism, including Bacteria and Archaea)
Taxonomy and Systematics (for publication of novel taxa, taxonomic reclassifications and reviews of a taxonomic nature)
Professional Development (including education, training, CPD, research assessment frameworks, research and publication metrics, best-practice, careers and history of microbiology)
If you are unsure which Section is most appropriate for your manuscript, for example in the case of transdisciplinary studies, we recommend that you contact the Editor-In-Chief by email prior to submission. Our scope includes any type of microorganism - all members of the Bacteria and the Archaea and microbial members of the Eukarya (yeasts, filamentous fungi, microbial algae, protozoa, oomycetes, myxomycetes, etc.) as well as all viruses.