Jens Gruhne

• Vicia faba leaves and calli were transformed using CRISPR Cas RNP. Two kinds of CPP fused SpyCas9 were used with sgRNA7, sgRNA5 or sgRNA13 targeting PDS exon 1, PDS exon 2 or MgCh exon 3 respectively. RNP were applied using high pressure spraying, biolistic delivery, incubation in RNP solution and infiltration of leaf tissue. A PCR and restriction enzyme based approach was used for detection of mutation. Screening of 679 E. coli colonies containing the cloned fragments resulted in detection of 14 mutations. Most of the 14 mutations were deletions of sizes 150, 500 or 730 bp. 5 out of the 14 mutations were point mutations located two to three bp upstream of PAM.