Establishment of a modified CRISPR/Cas9 system with increased mutagenesis frequency using the translational enhancer dMac3 and multiple guide RNAs in potato

CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5′-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.