Fig. 3

Identification of CRISPR/Cas9-induced crtiso mutants in tobacco by MSBSP-PCR. a Genomic DNA from T₀ plants was used for PCR amplification using primers CRTISO-F and CRTISO-R. The amplification products (with gDNA from a putative T₀ transgenic tobacco line as template) were cloned into a TA-vector and recombinant bacteria screened for the presence of mutations at the target site. The absence of amplification product when using the target specific primer CRTISO-T and CRTISO-R denotes the existence of a mutation (bacterium B3, B6 and B7 in lower panel). As a positive control bacterial clones were also amplified with CRTISO-F and CRTISO-R (upper panel). b Genomic DNA from T₀ tobacco transgenic lines was amplified by PCR with primers CRTISO-F and CRTISO-R (upper panel) and the amplification products subjected to a second PCR using CRTISO-T and CRTISO-R primers (lower panel). The absence of an amplification product in the second PCR suggests that the transgenic line is homozygous/biallelic (lower panel lane L4)