![]() PCR products from pPZP-SpCas12f Tub-1 or pPZP-SpCas12f Tub-2 transformed calli were cloned in E. The remaining percentage in each callus is the frequency of WT (no mutations). The number shown to the right side of each sequence is the frequency (ratio of the number of clones in which mutations were detected to total number of clones analyzed). The square box on the sequence indicates microhomology sequences located at the breakpoint. Blue and red letters on the sequence indicate the PAM sequence and the target sequence, respectively. (B) Examples of Sanger sequencing results in HMA positive samples on Tub-1 (upper panel) and Tub-2 (lower panel) in transformed calli. NT non-transformed, -g calli transformed with pPZPSpCas12f vector without sgRNA. The numbers 1-10 and 11-20 indicate representative HMA-negative and -positive samples, respectively. We picked up 10 HMA-negative samples and 10 HMA-positive samples from Supplementary Figure S1. SpCas12f is thus a useful tool for genome editing in rice and is suitable for virus vector-mediated genome editing due to its very small size.Īnalysis of SpCas12f-induced mutations in rice calli (A) Heteroduplex mobility assay (HMA) to detect mutations on Tub-1 (upper panel) and Tub-2 (lower panel) in transformed calli. Taken together, we demonstrated that SpCas12f can be used to achieve targeted mutagenesis in rice. This might be due to the culture conditions, with relatively higher temperature (30☌ or higher) and constant light during callus proliferation. Surprisingly, however, mutations can be introduced without heat-shock treatment in rice. In the previous report in maize, mutations were introduced by treatment with heat shock at 45☌ for 4 h per day for 3 days no mutations were introduced under normal growth conditions at 28☌. It was confirmed that the mutations in the regenerated plants were inherited to the next-generation. Furthermore, mutant plants were regenerated successfully from the mutated calli. Moreover, off-target mutations by SpCas12f were not found. Most mutation patterns were deletions, but base substitutions and insertions were also confirmed at low frequency. Detailed analysis by amplicon sequencing revealed estimated mutation frequencies (a ratio of the number of mutated calli to that of SpCas12f-transformed calli) of 28.8% and 55.6% in two targets. Molecular analysis of SpCas12f-transformed calli showed that mutations were introduced successfully into the target region. An expression vector encoding rice codon-optimized SpCas12f and sgRNA for OsTubulin as a target was introduced into rice calli by Agrobacterium-mediated transformation. In this study, we applied SpCas12f to genome editing in rice-one of the most important staple crops in the world. However, there are no reports of genome editing using SpCas12f in crops other than maize. Recently, targeted mutagenesis in human cells and maize using Cas12f derived from Syntrophomonas palmitatica (SpCas12f)-a very small Cas of 497 a.a, which is a more suitable size for virus vectors-was reported. Cas9 derived from Streptococcus pyogenes (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues).
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