Fig 1.
Generation of targeted mutations in callus tissues of potato using CRISPR/Cas reagents.
A. Target sites of single-guide RNA within potato StALS1 and -2 genes. A single nucleotide polymorphism (lowercase) exists in the gRNA746 target site of StALS2 but not gRNA751. AloI and BslI restriction enzyme sites exist in sgRNA target sites of both genes (underlined). Arrows indicate primers used for enrichment PCR and restriction enzyme digestion assays. PAM sequences are in gray. B. Modified enrichment PCR assay using potato callus tissue transformed with gRNA746 and gRNA751 CRISPR/Cas reagents. Total genomic DNA was subjected to PCR amplification of the StALS target site (bottom image; 448 bp), digested overnight with AloI (lanes 1, 3, 5, 7, 9, 11) or BslI (lanes 2, 4, 6, 8, 10, 12), and reamplified (top image; 448 bp) to generate an enriched amplicon. Enriched band intensities were normalized by dividing the quantified band intensity of the enriched band by the primary PCR amplicon (S1 Table). Positive (+), negative (-) and non-detectable (ND) enriched bands have normalized intensities equal or over 0.5, less than 0.5 and equal or more than 0.05, or less then 0.05, respectively. Diploid (X; lanes 1–6) and tetraploid (D; lanes 7–12) genotypes were tested using both sgRNAs in the conventional 35S (M; lanes 1, 2, 7, 8) and geminivirus LSL (L; lanes 3, 4, 9, 10) T-DNA backbones. Wild-type (wt; lanes 5, 6, 11, 12) genomic DNA was used as non-transformed controls.
Table 1.
Summary of targeted mutation screen of primary events and enrichment PCR results from callus.
Diploid (X914-10) and tetraploid (Désirée) genotypes were stably transformed with gRNA746 and gRNA751 CRISPR/Cas reagents in a conventional 35S or geminivirus LSL T-DNA backbone using hygromycin selection (Total events). A restriction enzyme digestion assay and quantification of resistant and digested bands were used to identify events with at least 1% mutation frequencies (# with mutations) and events above a threshold using expected single allele mutation frequencies (# above threshold) (S2 Table). Percentages are of total events and modified enrichment PCR results come from Fig 1B and S1 Table.
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.
Fig 3.
Inheritance of targeted mutations and Cas9 in progeny of primary CRISPR/Cas events.
Three primary events with cloned targeted mutations (lanes 1, 8 and 12; underlined) were used to generate genetic populations to assess inheritance of targeted mutations. The diploid event (lane 1; X46-3) was crossed to an inbred diploid line, M6 (lane 18) as the female parent while tetraploid events (lanes 8, 12; D46-44, D46-9) were selfed. Six progeny from the X46-3 population (lanes 2–7) and three progeny from the D46-44 (lanes 9–11) and D46-9 (lanes 13–15) populations were assessed for A) targeted mutations using a restriction digestion assay (top gel) and inheritance of Cas9 (bottom gel) and used for B) cloning targeted mutations using previously described methods (Fig 2 and S5 Fig). The PCR assay used for detecting Cas9 (A; bottom gel) produced a 1144 bp amplicon with each lane corresponding to the top gel and is further described in S6 Fig Wild-type Désirée and M6 were used as negative controls (lanes 16 and 18, respectively) and a 1:1 template mixture with wild-type and mutated DNA was used as a positive control (lane 17). The lengths of deletions (-) or insertions (+) of the targeted mutations in progeny (B) are in parenthesis to the left of each cloned mutation and the number of reads generated in the primary event (F0) or individual progeny (F1) are in brackets on the right. All targeted mutations were aligned to wild-type sequence and cloned from StALS1 unless indicated on the right. PAM sequences are in gray.
Table 2.
Summary of targeted mutation screen of progeny from primary events and inheritance of Cas9.
Progeny from diploid (X46-3) and tetraploid (D46-9, D46-44) primary events were screened for inheritance of Cas9 (# of Cas9-free progeny) and Cas9-free progeny were screened for targeted mutations (# of Cas9-free progeny with mutations) (S6 and S7 Figs). Mutation transmission percentages are of Cas9-free progeny with targeted mutations and percent of Cas9-free are of the number of progeny screened. Mutations detected are targeted mutations cloned from primary events (F0) and progeny (F1).