CRISPR-Cas technology: Targeted Genome Editing Technology

The interactions between prokaryotes and the viruses that infect them have evolved, leading to a wide diversity of CRISPR-Cas systems. CRISPR-Cas systems are generally divided into two categories (Class 1 and Class 2). To date, most researchers have used the Type 2 CRISPR-Cas system, and in this class, the most studied type II is the CRISPR-Cas9 system.

CRISPR/Cas9 system involves two essential components: target-specific CRISPR gRNA and Cas9 nuclease. In eukaryotic systems, CRISPR/Cas9 is used for genomic editing through specific targeting of DNA by sgRNA, a combination of the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA), mediated through base pairing over the ~20-nt guide sequence ^[1]. Cas9 recognizes a very short conserved sequence (a few nucleotides in length) adjacent to the guide sequence called the “protospacer adjacent motif” (PAM). Once directed to the DNA target site, Cas9 generates a double-strand break (DSB) that can be repaired either through the indel mutation-introducing non-homologous end-joining (NHEJ) or the high-fidelity homologous directed repair (HDR), resulting in gene knockout effects or template-dependent gene replacement.