Figure 3.

Improvement of mutation efficiency of Cas9/sgRNA in amphioxus. The induced mutation ratios shown under each gel image were estimated by either a T7 endonuclease assay (for mCherry-sgRNA) or a restriction endonuclease assay (for sgRNAs targeting the Dkk1/2/4, Nodal and Hwa genes). Red arrowhead indicates bands released by endonuclease digestions. PCR, PCR products not treated with the restriction enzyme; WT, digestion of PCR products from control (uninjected) embryos with the restriction enzyme. (A) Monomerization of sgRNAs (top) and its effect on mutation efficiency (bottom). sgRNA was monomerized using a denaturing step. B, before denaturation; A, after denaturation; D, dimer, M, monomer. (B) Comparison of mutation efficacies between version 1 (V1) and version 2 (V2) sgRNAs targeting the Nodal gene.