Three delivery methods of CRISPR/Cas9 to induce targeted mutations into centromeric histone H3 (CENH3) in carrots (Daucus carota L.)

The centromere-specific histone 3 (CENH3) in carrots (Daucus carota) was mutated using CRISPR/Cas9. Transformation with Agrobacterium tumefaciens and Agrobacterium rhizogenes resulted in transgenic lines with secondary mutations and lower levels of CENH3. Transient transformation of carrot protoplasts with CRISPR/Cas9 ribonucleoprotein complexes (RNPs) resulted in transgene-free and mutated protoplasts. The transgene-free method was identified as the preferred method due to the absence of secondary mutations.