An expanded toolkit for genome editing

CRISPR utilizes endonuclease proteins like Cas9 guided by engineered RNA to create breaks in target DNA. Each Cas9 ortholog only cuts targets near a particular sequence motif, such as Streptococcus pyogenes Cas9s NGG requirement. These requirements mean CRISPR lacks the flexibility of the older technologies. To increase available targets, four alternative nucleases were utilized. These are the Cas9s from S. pyogenes, Staphylococcus aureus, and Streptococcus thermophilus, and Cpf1 from Acidaminococcus spp. Nucleases were cloned into an expression cassette of native legume sequences and fitted with a novel nuclear localization signal from Glycine max. CRISPR nucleases from S. pyogenes, S. aureus, and S. thermophilus were confirmed to edit hairy root cultures. Hairy root editing efficiency is low with the plant-derived expression cassette, but is sufficient for editing in somatic embryos, as S. pyogenes has produced T0 plants with bi-allelic mutations.