How is CRISPR-Cas9 used in gene knockout and knock-in?

Gene knockout is a genetic technique which makes a gene permanently inoperative. CRISPR employs the non-homologous end joining (NHEJ) mechanism to disrupt the gene function via its error-prone nature. Nucleotides may be inserted or deleted between the broken ends, causing shifts in the reading frame of a gene and thus terminating the gene’s function.

The application of multi-guide sgRNA can also lead to a knockout, where multiple cuts are made in the DNA, inducing one or more large fragment deletion in the target gene. Since several amino acids are removed by these deletions, the targeted gene is likely to be completely inoperative.

Gene knock-in is the process replacing a gene with another, which can be achieved by inducing DNA repair via homology-directed repair (HDR) mechanism. For this purpose, a DNA template bearing the knock-in sequence flanked by regions of homology must be introduced into cells along with the CRISPR components. This method has been employed in a variety of genomic rewriting applications, from introducing single point mutations, inserting selectable markers, to even correcting a genetically-encoded mutation.