Suresh Ramakrishna1, Abu-Bonsrah Kwaku Dad1, Jagadish Beloor2, Ramu Gopalappa1, Sang-Kyung Lee2, and Hyongbum Kim1, 3
1Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul 133-791, Republic of Korea
2Dept of Bioengineering and Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul 133-791, Republic of Korea
RNA-guided endonucleases (RGENs) derived from the CRISPR/Cas system represent an efficient tool for genome editing. RGENs consist of two components: Cas9 protein and guide RNA. Plasmid-mediated delivery of these components into cells can result in uncontrolled integration of the plasmid sequence into the host genome and unwanted immune responses and potential safety problems that can be caused by the bacterial sequences, and requires transfection tools for mediating the delivery. Here we show that simple treatment with cell-penetrating peptide (CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs lead to endogenous gene disruptions in human cell lines. The Cas9 protein was conjugated to CPP via a thioether bond, whereas the guide RNA was complexed with CPP, forming condensed, positively charged nanoparticles. Simultaneous and sequential treatment of human cells, including embryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinoma cells, with the modified Cas9 and guide RNA leads to efficient gene disruptions with reduced off-target mutations relative to plasmid transfections, resulting in the generation of clones containing RGEN-induced mutations. Our CPP-mediated RGEN delivery process provides a plasmid-free and additional transfection reagent-free way to use this tool with reduced off-target effects. We envision that our method will facilitate RGEN-directed genome editing.