CRISPR/Cas9 technology has become the most efficient method for genome editing in many plant species, including important industrial crops such as potatoes. This study used three target regions (T1, T2, and T3) in gbss exon I, whose sequences were first inserted into the BbsI sites in the appropriate guide RNA (gRNA) vector (pEn-Chimera, pMR203, pMR204, and pMR205), and then localized between the AtU6 promoter and the gRNA scaffold sequence. Expression vectors were constructed by introducing gRNA genes into the pMR287 (pYUCas9Plus) plasmids using the MultiSite Gateway system by attR and attL sites. The three target regions of mutant potato lines were analyzed. The use of CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis allowed tri- or tetra-allelic mutant potato lines to be generated. Multiple nucleotide substitutions and indels within and around the three target sites caused a frameshift mutation that led to a premature stop codon, resulting in the production of gbss-knockout plants. Mutation frequencies and analysis of mutation patterns suggested that the stably transformed Cas9/multiple guide RNA expression constructs used in this study can induce targeted mutations efficiently in the potato genome. Full knockout of the gbss gene was analyzed by CAPS, Sanger sequencing and iodine staining. The present study demonstrated successful CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato gbss gene by Agrobacterium-mediated transformation, resulting in an amylose-free phenotype.

CRISPR/Cas9技术已成为许多植物物种基因组编辑最有效的方法，包括马铃薯等重要经济作物。本研究使用了gbss外显子 I中的三个目标区域（T1、T2 和 T3），其序列首先插入适当的引导 RNA (gRNA) 载体（pEn-Chimera、pMR203、pMR204 和 pMR205）中的BbsI位点，然后定位在 AtU6 启动子和 gRNA 支架序列之间。通过使用 MultiSite Gateway 系统通过 attR 和 attL 位点将 gRNA 基因引入 pMR287 (pYUCas9Plus) 质粒来构建表达载体。分析了突变马铃薯品系的三个目标区域。使用 CRISPR/Cas9 介导的多引导 RNA 靶向诱变可以产生三等位基因或四等位基因突变马铃薯品系。三个靶位点内部和周围的多个核苷酸取代和插入缺失导致移码突变，导致过早终止密码子，从而产生gbss敲除植物。突变频率和突变模式分析表明，本研究中使用的稳定转化的Cas9/多重指导RNA表达构建体可以在马铃薯基因组中有效诱导靶向突变。通过 CAPS、Sanger 测序和碘染色分析gbss基因的完全敲除。本研究证明，通过农杆菌介导的转化，在马铃薯gbss基因中成功实现了 CRISPR/Cas9 介导的多引导 RNA 靶向诱变，从而产生了无直链淀粉的表型。