Suresh Ramakrishna1, Abu-Bonsrah Kwaku Dad1, Jagadish Beloor2, Ramu Gopalappa1, Sang-Kyung Lee2 and Hyongbum Kim1
- Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul 133-791, Republic of Korea
- Department 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. Furthermore, this delivery method requires transfection tools. Here we show that simple treatment with cell-penetrating peptide (CPP)–conjugated recombinant Cas9 protein and CPP-complexed guide RNAs leads 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 method to use this tool with reduced off-target effects. We envision that our method will facilitate RGEN-directed genome editing.
CovalX Technology Used (Click each option to learn more)
A sequence in which Cas9 was encoded with a cysteine at the C terminus was created by using PCR amplification of a prior Cas9 plasmid as the template. It was then cloned into the pET28-(a) vector that has a His-tag encoding sequence at the N terminus. With the pET28-(a) vector encoding Cas9, E.coli BL21 cells were transformed overnight at 30 °C with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The sample was centrifuged at 20,000g for 20 minutes at 4 °C for cell collection. Then lysed in a lysis buffer (20 mM Tris-Cl (pH 8.0), 300 mM NaCl, 20 mM imidazole, 1 x protease inhibitor cocktail, 1 mg/mL lysozyme) using sonication (40% duty, 10 sec pulse, 30 sec rest, 10 minutes total, on ice) in order to induce protein expression. The Cas9 protein was purified using a column that contained Ni-NTA agarose resin as well as an AKTA prime instrument at 4 °C. Following purification, the column-bound protein was extracted using an elution buffer (20 mM Tris-HCl (pH 8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% glycerol). The purified proteins were diluted in distilled water and then concentrated before being mixed with DHB matrix solution (10 mg/mL; 1:2, v/v) and dropped onto the MALDI plate. Analysis of the plate was performed on a mass spectrometer that had been modified with a CovalX HM2 detection system and calibrated using the CovalX C150 Calibration Kit.