Efficiency of potato genome editing: Targeted mutation on the genes involved in starch biosynthesis using the CRISPR/dMac3-Cas9 system

Potato (Solanum tuberosum L.) has a tetraploid genome. To make mutant lacking specific gene function, it is necessary to introduce mutations into all four alleles. achieve this goal, we developed powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased of downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) elevated frequency generation rate mutation. This enabled create 4-allele mutants granule-bound starch synthase (GBSS) and branching enzyme (SBE). These indicated functionally defective features, suggesting succeeded in efficient potato Here, show effect number gRNAs for mutagenesis target using GBSS1 gene. gRNA genes achieved higher mutation efficiency than with two gRNAs, being influenced by dose at region. alleles SBE3 contained SNPs caused sequence differences but these functioned efficiently. However, many rearrangement events large deletions were induced. results support importance accurate binding sequence, may lead hint avoid unexpected on off-target sites.