Genome editing using CRISPR technologies

Use of CRISPR (clustered regularly interspaced short palindromic repeats) and associated Cas enzymes for genome editing has been a major technological breakthrough, making genome modification in cells or organisms faster, more efficient, and more robust than previous genome editing methods.

The most commonly used system combines a guide RNA with the Cas9 enzyme from Streptococcus pyogenes to achieve gene disruption or modification via knock-in or knock-out strategies (see below).

Other approaches exist, making use of e.g. modified Cas9 variants (nickases, dead variants, Cas9 fusions to other DNA modifying enzymes). In addition alternative Cas enzymes are available, such as Cas12a (also termed Cpf1) from Acidaminococcus sp. BV3LC, which allow targeting of gene locations unaccessible to the Cas9 system.

The Cas enzyme combines with a guide RNA to form an editing complex, the so-called Ribonucleoprotein (RNP). The role of the guide RNA is to guide the editing complex to the appropriate location in the genome. Recognition of the genomic region to be targeted is achieved by complementarity between the variable region of the guide RNA and the genomic sequence. The target region must be adjacent to a so-called PAM (protospacer adjacent) motif. For Cas9 the PAM motif is NGG, for Cas12a it is TTTV . Once bound to the target sequence, the Cas enyzme will effect a double strand cut.

Once Cas9 complexed to a guide RNA is delivered into the cells it homes in on the genomic target sequence and performs a double-strand cut. The cell attempts to repair the cut and wo outcomes are possible:

Repair via non-homologous end joining (NHEJ), resulting in a knock-out

The two strands are «patched together» by enzymes of the NHEJ pathway. This often results in small deletions or insertions (indels) which have the potential to disrupt the open reading frame of the target gene, leading to a knock-out of gene expression. This approach typically yields high editing efficiencies.

Repair via the HDR pathway, resulting in a knock-in

If a DNA donor template is supplied along with the CRISPR components, knock-in of a desired sequence can be achieved via the homologous directed repair (HDR) pathway. The donor template contains the sequence of interest (knock-in sequence) flanked by sequences that are homologous to the target genomic sequence (homology arms). Because a defined template is used, HDR repair is typically accurate and the sequence of interest is inserted into the genome seamlessly. However, HDR only occurs in the S and G2 phases of the cell cycle and is less efficient than NHEJ. Efficiencies around 20% may be achieved but the outcome strongly depends on the cell type used.