4.7. CRISPR/Cas9 Vector Construction and Genotyping Analysis

The P201N-Cas9 vector was used to construct a plasmid vector expressing Cas9 and sgRNA simultaneously in lemon. The Cas9 gene was codon-optimized for dicotyledons and placed downstream of the StUbi-3 promoter together with customized sgRNA driven by the MtU6 promoter. We designed the sgRNAs using the web-based tool CRISPR-P2.0 [80] and used CRISPR web tool to predict the off-target sites [81]. The primers used for detection vector construction, sequencing and CiMADS43 genotyping are all displayed in Table S4.