Figure 2. Targeted mutagenesis in cotton protoplasts.

(A) Detection of mutations at the GhCLA1 loci. (B) Detection of mutations at the GhVP loci. Detection of mutations using RE-PCR assay and sequence of the CRISPR/Cas9 system-induced mutation in cotton protoplasts. DNA fragments obtained by PCR amplification of genomic DNA from control protoplasts either undigested (lane 1) or digested (lane 2) with PstI (A) or HinfI (B). Genomic DNA from transgenic protoplasts were digested before PCR amplification with PstI (A) or HinfI (B) and, therefore, PCR products were almost exclusively products (lane 4) from mutated DNA sequences. Sequences of the mutations induced by the CRISPR/Cas9 system are shown at the bottom. The change in the number of nucleotides is shown to the right of each sequence. +: insertion; −: deletion; S: substitution.