Overexpression and cell-penetrating peptide-mediated delivery of Cas9 and its variant(s) for targeted genome editing.

Abstract

The clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated (Cas) system is a powerful tool that has been used for genome editing. Cas9 is a bacterial RNA-guided endonuclease that cleaves target DNA and modifies a cell's genome. Despite the advantages and promising results of the use of CRISPR-Cas9 as a molecular tool and potential therapeutic protein, the production and purification of the recombinant Cas9 protein remain a challenge. Following protein expression and purification, delivering the Cas9 protein into the cells is a key concern for many researchers. Typically, Cas9 and gRNA are delivered by using either plasmid or viral vectors. However, the plasmid or viral-mediated delivery triggers an undesirable immune response and leads to uncontrolled integration of the plasmid into the host genome. We envision that direct delivery i.e. conjugating the protein with cell-penetrating peptide (CPP) will lead to endogenous gene editing with reduced off-target effects when compared to the other delivery methods that require a transfection reagent. Our study aims to determine if the vector strains or culture conditions need to be optimized based on different Cas9 variants. Additionally, this study intends at delivering Cas9 derived from Streptococcus pyogenes (SpCas9) and other Cas9 variants into the mouse fibroblasts (NIH3T3 cell line) by conjugating the protein with CPP through thiol chemistry. Therefore, here, we methodically evaluate the expression of recombinant SpCas9 and other Cas9 variants in four different E. coli strains (Rosetta, BL21 (regular), BL21 PlysS, and BL21 star). After optimization of the conditions (temperature and post-induction time), BL21 (DE3) PlysS strain efficiently expressed the SpCas9 protein. Overall, the results of this study will inform optimized methods for CPP-mediated delivery of Cas9.