Plasmid-based and -free methods using CRISPR/Cas9 system for replacement of targeted genes in Colletotrichum sansevieriae

Abstract

The CRISPR-Cas9 system has a potential for wide application in organisms that particularly present low homologous integration rates. In this study, we developed three different methods using this system to replace a gene through homology-directed repair in the plant pathogenic fungus Colletotrichum sansevieriae, which has a low recombination frequency. The gene encoding scytalone dehydratase was used as the target so that mutants can be readily distinguished owning to a lack of melanin biosynthesis. First, we performed a plasmid-based method using plasmids containing a Cas9 expression cassette and/or a single-guide RNA (sgRNA) under the control of the endogenous U6 snRNA promoter, and 67 out of 69 (97.1%) transformants exhibited a melanin-deficient phenotype with high efficiency. Second, we performed a transformation using a Cas9 protein/sgRNA complex and obtained 23 out of 28 (82.1%) transformants. Lastly, we developed a hybrid system combining a Cas9 protein and donor DNA-sgRNA expression plasmid, which yielded 75 out of 84 (89.2%) transformants. This system was also applicable to four other genes at different loci of the fungus. This is the first study to establish a CRISPR/Cas9 gene replacement system in Colletotrichum spp. and it presents a potential application for a broad range of use in other species of the genus.

Current Techniques to Study Beneficial Plant-Microbe Interactions

“…The strains with those mutations were then tested for their ability to function, thereby proving their involvement in specific pathogen activities. Thus, the CRISPR/Cas system has been utilized in the mutagenesis of Fusarium oxysporum , Fusarium proliferaturm , Alternaria alternata , Phytophthora spp., Sclerotinia sclerotiorum , and Colletotricheum sansevieriae [ 199 , 200 , 201 , 202 , 203 ]. These experiments were performed as a means of better understanding the pathogen infection process.…”

Harnessing the Potential of CRISPR/Cas in Atherosclerosis: Disease Modeling and Therapeutic Applications

“…The two components can be delivered into the cells in different forms such as plasmids, mRNAs, and RNP. Plasmid-based method utilize plasmids containing expression cassettes for Cas9 and guide RNA, and the expression of the two components are controlled by the endogenous U6 promoter [93]. Besides, mRNA for Cas9 and guide RNA can be delivered into target cells simultaneously to achieve genome editing [94].…”

“…Besides, mRNA for Cas9 and guide RNA can be delivered into target cells simultaneously to achieve genome editing [94]. Next, the plasmid-free method emphasized on the formation of RNP complexes before being introduced into the cells for genome editing [93]. The RNP approach was found to exhibit higher editing efficiency with lower off-target effects in hard-to-transfect cells [95].…”

Applications of CRISPR technology in studying plant-pathogen interactions: overview and perspective

“…The CRISPR/SpCas9-mediated genome editing technology has been successfully established in a wide range of fungal species, including Magnaporthe oryzae (Arazoe et al 2015;Foster et al 2018 (Li et al 2018), Colletotrichum sansevieriae (Nakamura et al 2019), B. cinerea (Leisen et al 2020), Sporisorium scitamineum (Lu et al 2017), Ustilaginoidea virens (Liang et al 2018), Ustilago maydis (Schuster et al 2016) and Ustilago tricophora (Huck et al 2019). In fungi, both SpCas9 and the sgRNA can be stably or transiently expressed by polyethylene glycol (PEG)-mediated transformation, Agrobacteriummediated transformation, electroporation, and biolistic transformation (Schuster and Kahmann 2019).…”