CRISPR-Cas9 Genome Editing Utilizing Chemically Synthesized RNA

The CRISPR-Cas9 system permits researchers to quickly edit genes for functional protein knockout in mammalian, fish and plant genomes, among others, and consequently has dramatically transformed biological research. The CRISPR-Cas9 system requires exogenous Cas9 nuclease to be delivered into the cell, which can be accomplished through transfection of an expression plasmid, mRNA or protein, or through transduction with lentiviral particles. Besides Cas9 nuclease, the natural CRISPR-Cas9 system also requires two RNA components: CRISPR RNA (crRNA) comprised of spacer-derived sequence and of repeat-derived sequence and tracrRNA, which hybridizes to the crRNA through repeat-derived sequences. The crRNA:tracrRNA complex recruits the Cas9 nuclease and cleaves DNA upstream of a protospacer-adjacent motif (PAM). The crRNA and tracrRNA can be linked together with a loop sequence for generation of a chimeric single guide RNA (sgRNA). In a vector-based approach, cloning and sequence verification of each sgRNA vector can be laborious and time consuming, especially if the goal is to study tens or thousands of gene targets. Likewise, in vitro transcription of the sgRNA also requires additional time and quality control to ensure consistency in length and purity of the transcribed product. In contrast, chemical synthesis can easily be employed for rapidly generating the crRNA and tracrRNA molecules separately or a synthetic sgRNA for direct delivery into cells for gene editing.