Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroomPleurotus ostreatus

Abstract

CRISPR/Cas9 has potential for efficient molecular breeding. Recently, a foreign-DNA-free gene-targeting technology was established by introducing a pre-assembled 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.

The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments

“…For example, the appropriate promoters for the expression of Cas9 and gRNA must be chosen, the codons of Cas9 must be optimized to ensure efficient translation into protein, which requires a selective marker for cytoplasmic maintenance, and fragmented plasmid DNA may be randomly integrated into chromosomal DNA. In this regard, directly introducing the Cas9-gRNA RNP complex to protoplasts has been attempted for gene editing in mushrooms, such as S. commune 27 , C. cinerea 28 , and P. ostreatus 29 , 34 .…”

“…Lastly, the random insertion of DNA fragments into the DSB site suggests that insertion of a specific DNA fragment can be facilitated when the RNP is transformed together with a DNA fragment with a specific function without using HR. This type of RNP-mediated gene editing and subsequent positive selection was employed in the double-gene targeting of P. ostreatus , in which both pyrG and fcy1 (encoding cytosine deaminase) were disrupted by a single transformation of Cas9/ pyrG sg1 and Cas9/ fcy1 sg2 RNPs and selected against 5-FOA and 5-fluorocytosine (5-FC) 34 . Deamination of 5-FC by fcy1 produces 5-FU which is the same toxic product produced by pyrG on 5-FOA.…”

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences

“…However, at present, the efficiency of genome editing using RNP-based methods may be lower than that of DNA-based methods (Boontawon et al 2021b ). The major challenge of the RNP-based method is the lack of an efficient selection system; this method has been applied only to a special target gene whose disruption makes it possible to screen for, such as pyrG (Boontawon et al 2021b ; Kowalczyk et al 2021 ; Eom et al 2023 ) or fcy1 (Boontawon et al 2023 ), unless a foreign selection marker was also introduced (Jan Vonk et al 2019 ; Boontawon et al 2021b ; Kowalczyk et al 2021 ; Pareek et al 2022 ). To overcome this problem, RNP-dependent CRISPR/Cas9 with two target genes, one for screening and the other the gene of interest, was developed in P. ostreatus (Boontawon et al 2023 ).…”

“…However, in G. lucidum , frequent integration of contaminated and mitochondrial DNA fragments at the target site has been observed in RNP-dependent genome editing (Eom et al 2023 ). Integration of unexpected DNA sequences at the target and off-target sites could be a fatal problem in CRISPR/Cas9, both in DNA- and RNP-dependent protocols, although successful isolation of small insertion/deletion mutations has been reported in P. ostreatus (Boontawon et al 2023 ).…”

Current Advances in the Functional Genes of Edible and Medicinal Fungi: Research Techniques, Functional Analysis, and Prospects

“…This resulted in editing efficiencies of up to 5% and the more rapid growth of transformants compared to common gene-editing techniques. In 2023, Boontawon et al [94] established a system of RNPs in P. ostreatus without exogenous DNA genes, and double-gene-edited mutants were obtained by screening RNP-added strains for 9-FOX resistance.…”