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Fig. 5 | Plant Methods

Fig. 5

From: A simple and cost-effective method for screening of CRISPR/Cas9-induced homozygous/biallelic mutants

Fig. 5

Identification of the CRISPR/Cas9-induced mutants in T1 tobacco plants by MSBSP-PCR. A first PCR reaction was conducted with genomic DNA isolated from T1 transgenic plants using locus primers PVY-F and PVY-R (upper panel) and the amplification products used as template for a secondary PCR with the target specific primer PVY-T and PVY-R (middle panel). Lanes 4-6 were identified as homozygous/biallelic mutants based on the absence of amplification product. To confirm the results of the MSBSP-PCR, the products of the first PCR were digested with PvuII (lower panel). Complete digestion of the amplification product indicates a WT gene sequence (WT lane); lanes 1-3 show a combination of digested and undigested product indicating that the plants were heterozygous. The undigested products in lines 4-6 identify the plants as homozygous or biallelic mutants

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