Figure 1.

Overview of the current Cas9-based gene editing systems. (A) When bound to a sequence-specific guide RNA (gRNA), the wild-type Cas9 recognizes a target sequence and induces a double-stranded break (DSB). The cell then employs one of its major DNA repair mechanisms, either Non-Homologous End Joining (NHEJ) or Homology-Directed Repair (HDR), to repair the DSB. NHEJ can result in random insertion/deletion mutations that disrupt the target gene, while HDR in the presence of a donor DNA molecule results in the integration of the donor DNA. (B) Two different Cas9 nickases (Cas9n), the Cas9 D10A and Cas9 H840A, have mutations in either the RuvC or HNH domains, respectively, and induce a single-stranded break in the target or non-target DNA strand, respectively. (C) The two types of single-base editors utilize a catalytically dead or nickase Cas9 fused with a deaminase enzyme. The cytidine deaminase-based editor can convert the C:G base pair into a T:A base pair, while the adenine deaminase-based editor can convert a T:A base pair into a C:G base pair. The cytidine deaminase-based editor has a uracil glycosylase inhibitor (UGI) directly connected to the C terminus of Cas9, which prevents the repair of U:G mismatches back into C:G base pairs. (D) The prime editor is the latest engineered Cas9 variant that is fused with reverse transcriptase (RT). The prime editing guide RNA (pegRNA) has two extended sequences, one to bind the nicked DNA strand (primer binding site) and another to serve as a template for reverse transcription. The RT converts a portion of the pegRNA into the DNA template that will then be integrated into the target DNA.