The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers’ ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.
The efficient editing of target genes in transgenic plants can provide researcher with desired mutants, which will accelerate the progress of gene function dissection. We confirmed that the sgRNAs complying with the criteria were efficient for target gene editing. Both this work and Ma et al.’s9 confirmed that at least two same snoRNA promoters can be used simultaneously to drive different sgRNAs. Our work also revealed that the number of sgRNA cassettes has no effect on the editing efficiency of sgRNAs. It is noteworthy that up to 84.8% of edited plants contained loss-of-function gene mutations (i.e., biallelic or homozygous mutations) in the T0 transgenic plants and they can be used directly for functional analysis. In our two-sgRNA expression plants, both targets sites can be edited simultaneously, which also caused deletion or inversion of DNA fragment between two target sites. The editing of both target sites will facilitate the gene correction via homologous recombination by providing a mutant plant with wild type DNA fragment donor. Altogether, our toolbox for sgRNA design criteria and assembly of multiplex CRISPR/Cas9-sgRNA system provides researchers with a new approach to efficiently edit one or multiple target sites and perform genetic improvement.
https://archive.is/AntWC