Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroom Pleurotus ostreatus
Issued Date
2023-01-17
Resource Type
eISSN
15746968
Scopus ID
2-s2.0-85151045039
Pubmed ID
36812945
Journal Title
FEMS microbiology letters
Volume
370
Rights Holder(s)
SCOPUS
Bibliographic Citation
FEMS microbiology letters Vol.370 (2023)
Suggested Citation
Boontawon T., Nakazawa T., Choi Y.J., Ro H.S., Oh M., Kawauchi M., Sakamoto M., Honda Y. Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroom Pleurotus ostreatus. FEMS microbiology letters Vol.370 (2023). doi:10.1093/femsle/fnad015 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/81683
Title
Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroom Pleurotus ostreatus
Other Contributor(s)
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.