Figure 2.

Tagging of endogenous gene is improved with MRN recruitment by chimeric Cas9. (A). Schematic representation of targeted PSMB6 locus. Stop codon is indicated in red. Guide sequence is in bold, with PAM sequence in pink (note that the guide is the minus strand). The region of the ssODN template encoding the Flag tag is shown. The left and right homology arms are 58 and 53 bp, respectively. (B). Insertion of Flag tag at C-terminus of PSMB6 is more efficient with MRN-recruiting Cas9 construct. HEK293 cells were transfected with the indicated amounts of Cas9/sgRNA encoding plasmids and with ssODN. Cells were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.