Using Staphylococcus aureus Cas9 to Expand the Scope of Potential Gene Targets for Genome Editing in Soybean

• Published in: International journal of molecular sciences Vol. 23; no. 21; p. 12789
• Main Authors: Zhang, Yan, Cai, Yupeng, Sun, Shi, Han, Tianfu, Chen, Li, Hou, Wensheng
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 24.10.2022; MDPI
• Subjects: Amino acids; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; CRISPR/Cas9; Editing; Efficiency; Gene deletion; gene editing; Gene Editing - methods; Genes; Genetic engineering; Genome editing; Genomes; Glycine max; Glycine max - genetics; Glycine max - metabolism; Hairy root; Mutagenesis; Mutation; Mutation rates; PAM; Proteins; soybean; Soybeans; Staphylococcus aureus; Staphylococcus aureus - metabolism; Staphylococcus aureus Cas9; Streptococcus pyogenes; Staphylococcus aureus Cas9; soybean; CRISPR/Cas9; PAM; gene editing
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms232112789

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a revolutionary genome editing technology that has been used to achieve site-specific gene knock-out, large fragment deletion, or base editing in many plant species including soybean ( ). The Cas9 (SpCas9) is widely used in plants at present, although there are some reports describing the application of CRISPR/Cpf1 in soybean. Therefore, the selection range of PAM (protospacer adjacent motif) sequences for soybean is currently limited to 5'-NGG-3' (SpCas9) or 5'-TTTN-3' (Cpf1), which in turn limits the number of genes that can be mutated. Another Cas9 enzyme from (SaCas9) recognizes the PAM sequence 5'-NNGRRT-3' (where R represents A or G), which can provide a wider range of potential target sequences. In this study, we developed a CRISPR/SaCas9 system and used this tool to specifically induce targeted mutations at five target sites in the (Glyma.16G150700) and (Glyma.16G044100) genes in soybean hairy roots. We demonstrated that this tool can recognize the PAM sequences 5'-AAGGGT-3', 5'-GGGGAT-3', 5'-TTGAAT-3', and 5'-TAGGGT-3' in soybean, and it achieved mutation rates ranging from 34.5% to 73.3%. Our results show that we have established a highly efficient CRISPR/SaCas9 tool that is as suitable as SpCas9 for genome editing in soybean, and it will be useful for expanding the range of target sequences for genome editing.