Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System
Fig 2
Generation and cloning of targeted mutations in primary events of potato using CRISPR/Cas reagents.
A. Restriction enzyme digestion assay of diploid (X; lanes 2–4) and tetraploid (D; lanes 5–8) primary events. Total genomic DNA from primary events was subjected to PCR amplification of the StALS target site and digested overnight with AloI yielding a 448 bp resistant band and 326 bp and 122 bp digested bands. Wild-type X914-10 (WT; lane 1) and Désirée (Fig 3A) genomic DNA were used as a negative controls. B. Cloned targeted mutations in primary events of potato. Diploid (X) and tetraploid (D) events constitutively expressing gRNA746 (46) and gRNA751 (51) CRISPR/Cas reagents were used for cloning. Resistant bands from restriction enzyme digestion assays were excised from 2.0% agarose gels, purified, and subcloned for Sanger sequencing. Sanger reads from each event were aligned to StALS1 and -2 wild-type sequence (WT) from each sgRNA target site (gRNA746; top alignments, gRNA751; bottom alignments). The lengths of deletions (-) or insertions (+) are in parenthesis to the left of each cloned mutation and the number of reads generated in the primary event (T0) or first clonal generation (CG1) are in brackets on the right. All targeted mutations were cloned from StALS1 unless indicated on the right. PAM sequences are in gray.