Generating mutants using CRIPR/Cas technology in Arabidopsis

Benefit description

In gene editing projects using CRISPR-Cas9, the following stages would be included: STAGE 1. Bioinformatic analysis of the target gene or genes. Selection of the zones of the sequence adjacent to the PAM sites, with better statistics and that do not generate "run-off" mutations in other genes of the same organism. Search for restriction targets that help genotyping. Use of CRISPR-Plant platforms and others for the design of guiding RNAs. Choice of the type of vector (germline, U6) depending on the objective of the project. STEP 2. Cloning of the target sequences in the target vector using the Golden Cloning technique. Transformation into Agrobacterium tumefaciens. STAGE 3. Stable transformation. STAGE 4. Selection of transformed and positive lines for Cas9. Genotyping using restriction enzymes and sequencing of target sequences. STAGE 5. Segregation of the different transgenic lines (T1 to T3). Genotyping by PCR and sequencing.