Targeted gene editing by transfection of in vitro reconstituted Streptococcus thermophilus Cas9 nuclease complex

Cas9 protein of the Type II CRISPRCas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) bacterial adaptive immune system emerged recently as a promising tool for genome editing in human and other eukaryotic cells. Cas9 binds a dual crRNA (CRISPR RNA)-tracrRNA (transactivating RNA) molecule, or an artificial single-guide RNA (sgRNA) into a functional complex that acts as an RNAdirected DNA endonuclease. It locates and binds to the target site guided by crRNA (sgRNA) while the Cas9 protein cuts DNA generating a double strand break (DSB) within the target sequence. In eukaryotic cells, DSB is repaired by “error prone” non-homologous end joining (NHEJ) or by homology directed repair (HDR) mechanisms resulting in the genome modification or insertion of new genetic information. Cas9 of Streptococcus pyogenes (SpCas9) is currently used as a model system for genome editing applications. Typically, the DNA expression cassettes encoding nucleus-targeted codon-optimized Cas9 protein and sgRNAs are transfected into the cells. The efficiency of DNA cleavage by plasmid-delivered Cas9 in eukaryotic cells depends on multiple factors, including expression vector design, transfection efficiency, cell type, recovery yield of functional Cas9 complex, and usually requires optimization of a set of experimental conditions. Cas9 delivery by plasmid transfection is still difficult to achieve for some hard-to-transform cell lines including human primary cells and pluripotent stem cells. Moreover, plasmid transfection occasionally results in undesirable integration of vector plasmid into the genome and is often inefficient and stressful to cells. Here we report an alternative way for the Cas9-mediated genome modification in eukaryotic cells (Fig. 1A) by chemical transfection of in vitro reconstituted functionally active Cas9-crRNA-tracrRNA complex of Streptococcus thermophilus (StCas9) CRISPR3-Cas system. The StCas9 protein bearing the nuclear localization signal (NLS) and 6xHis tag was purified from E.coli, and the StCas9 complex was reconstituted in vitro as described by Karvelis et al. To enable the delivery of reconstituted StCas9 complex into CHO-K1 cells, transfection experiments were performed using a protein delivery agent TurboFect. Alternatively, other transfection reagents like Lipofectamine 2000 or 3000 can be used to transfect Cas9 complexes into cells (data not shown). StCas9 localization was monitored using mouse polyclonal antiCas9 antibodies along with FITC-labeled secondary antibodies; crRNA-tracrRNA duplex was detected via 30-biotin labeled tracrRNA using streptavidin-coupled Qdots 585 nm. Both StCas9 and tracrRNA were observed in the perinuclear region and within the nucleus indicating that in vitro pre-assembled StCas9 complexes can be efficiently delivered into mammalian cells for targeted genome modification (Fig. S1). To monitor the DNA cleavage activity of transfected Cas9 complexes in mammalian cells, we constructed a dual reporter cassette bearing Red Fluorescent Protein (RFP) and enhanced Green Fluorescent Protein (eGFP) genes (Fig. 1B). eGFP gene contains 2 sites, T1 and T2, targeted by 2 different StCas9 complexes. The I-CreI nuclease target site was also engineered into the cassette. In the absence of Cas9, eGFP fluorescence should be observed following intron processing in vivo. Cas9 facilitated DSB at T1 or T2