CRISPR/Cas9

CRISPR/Cas9 (Clustered Regularly-Interspaced Short Palindromic Repeats/Cas9) is a bacterial immune system that has been adapted for genome editing in mammalian cells. Cas9 is a programmable nuclease that generates double-stranded breaks (DSB) in DNA dictated by binding of a ~20 nucleotide recombinant “guide RNA” (gRNA) to the target site. DSB’s produced by Cas9 are most often repaired through the cell’s error-prone non-homologous end joining (NHEJ) pathway, resulting in small insertions or deletions (indels). The vast majority of indels shift the reading frame, introducing a premature stop codon or resulting in nonsense mediated decay of the mRNA-effectively “knocking out” a gene. The CRISPR/Cas9 system has substantially advanced efforts in specific gene editing and has been successfully applied to modify the episomal genomes of human and other organisms. The CRISPR/Cas9 system utilizes a prokaryotic RNA-guided programmable nuclease that can make a double-strand DNA break (DSB) at a specific site under the guidance of a leading RNA. This DSB process depends on the co-expression of two basic components: a guide RNA (gRNA) and Cas9 nuclease. Making a specific DSB can trigger DNA repair via either error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR). In the presence of the CRISPR/Cas9 system, the NHEJ inhibitor SCR7 is proven to increase the efficiency of Cas9-mediated HDR by at least by 7-fold in mammalian cells. Genome editing via CRISPR/Cas9 has become an efficient and reliable way to make precise, targeted changes to the genome of living cells.