Highly efficient CRISPR/Cas9-RNP mediated CaPAD1 editing in protoplasts of three pepper (Capsicum annuum L.) cultivars

Figures & data

Figure 1. In vitro cleavage assay for CRISPR/Cas9 RNP-mediated CaPAD1 editing in three pepper cultivars.

(a) The overview of CaPAD1 genomic locus. The five designed guide RNAs (sgRNA1–5) are marked with red triangles at three exons. The genomic loci of Dempsey, C15, and Younggo 4 were sequenced by Sanger sequencing, (b) Target sequences of the five sgRNAs. Red, PAM sequence (NGG) (c, d, e) In vitro cleavage assay with preassembled Cas9-only as a control and 5 Cas9-sgRNAs for the CaPAD1 gene in Dempsey, C15, and Younggo 4.

Figure 2. Three in vitro cultured pepper cultivars and RNP-delivered pepper protoplasts.

(a) Dempsey, (b) C15, and (c) Younggo 4. For each cultivar: 5–6 weeks old pepper cultivars (left), protoplasts immediately after PEG-mediated transfection (center), and protoplasts 48 h after incubation (right). The harvested pepper protoplasts for analyzing the CaPAD1 editing were all viable and without any cellular damage before/after CRISPR/Cas9 RNP delivery. White-scale bars, 1 cm; black-scale bars, 100 µm.

Figure 3. Analyses of CRISPR/Cas9 RNP-mediated CaPAD1 editing in three pepper cultivars.

(a, c, e) Indel frequencies of Dempsey, C15, and Younggo 4. The indel frequency (%) was calculated by dividing the number of sequencing reads containing an indel at the target site by the number of total sequencing reads. The data are presented as the mean of at least five biological replicates with standard deviation. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant (p > 0.05). Statistical significance was evaluated using one-way analysis of variance. (b, d, f) Indel patterns of the three CRISPR-edited cultivars: Dempsey, Younggo 4, and C15. Red, PAM sequence; blue, Cas9 target sequence; red dashed line (-), deleted nucleotide; red small letter, inserted nucleotide.

Figure 4. In vivo analyses of CaPAD1-sgRNA2 or CaPAD1-sgRNA5 activities at potential off-target sites across three pepper genomes.

(a) Off-target (OT) analyses of Dempsey, indel frequencies (%) at 11 potential OTs relative to the CaPAD1sgRNA2 of Dempsey genome were evaluated in Cas9-sgRNA2 RNP complexes delivered Dempsey protoplasts compared to Cas9 only by targeted deep sequencing analyses (n ≥ 3). (b) C15, (c) Younggo 4, indel frequencies (%) at 5 OTs relative to the CaPAD1sgRNA5 of C15 or Younggo 4 genomes were evaluated in Cas9-sgRNA5 RNP complexes delivered C15 or Younggo 4 protoplats by targeted deep sequencing analyses (n ≥ 3). No mutations were detected at any of the OT loci. Red, PAM sequences; blue, mismatched nucleotide bases.