In silico design of sgRNA for CRISPR/Cas9-mediated FaRALF33 gene mutagenesis to decrease the infection process to Colletotrichum acutatum in strawberry

Abstract

Rapid alkalinization factors (RALFs) are ubiquitous cysteine-rich peptides present in plants. They exert diverse functions as hormonal signals in various processes, including cell growth, root elongation, and fertilization. RALF peptides can also act as negative regulators of the plant immune response, inhibiting the formation of the signal receptor complex for immune activation. In Fragaria × ananassa, silencing of FaRALF33 gene plays a key role in the defense against the fungal pathogen Colletotrichum acutatum. In this study, single-guide RNA (sgRNAs) were designed in silico for clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) 9-mediated FaRALF33 gene mutagenesis in F. × ananassa for the reduction of C. acutatum infection. FaRALF33 was compared with homologous RALF33 sequences from other plant species, showing that the amino acid sequence of FaRALF33 presents typical sequences of known RALF peptides in RRILA proteolytic site, in addition to tight clustering presented by FaRALF33 with FvRALF33. The online tool CHOPCHOP provided 73 hits for FaRALF33 gene, selecting two sgRNA sequences for mutagenesis, sgRNA 1 (5’-CGACTCTCCCATCTCTTGGACT-3’) and sgRNA 2 (5’-GCAAGCAACGGCAGCGATCA-3’). The predicted secondary structures of the selected sgRNAs presented efficient structures in targeted mutagenesis. The pCas9-TPC-GFP-2XsgRNA vector for CRISPR/Cas9-mediated FaRALF33 gene mutagenesis was designed in silico with two sgRNA sequences (with Arabidopsis thaliana U6-26 promoters) and a green fluorescent protein marker.