Advancing Precision Medicine: Recent Innovations in Gene Editing Technologies

Figure 1. Nickase‐based approach for the deletion and integration of genes. A) Prime editing: This design employs pegRNAs that target DNA strands. The pegRNA specifies the sites to be cut and the 3′ flap that contains the edited sequence integrated into the DNA by the DNA repair mechanism. The regions where DNA is edited are indicated by blue in the 3′ flap regions of homology shown in green and red boxes (red to red and green to green). B) PRIME DEL: This design employs pairs of pegRNAs that target opposite DNA strands. Each pegRNA specifies the sites to be cut at both ends of the desired deletion and includes a 3′ flap that complements the region targeted by the other pegRNA. The regions where DNA is excised are indicated by shaded gray boxes, with the 3′ flap regions of homology shown in green and red (red to red and green to green). C) Twin Editing: This method allows for the flexible deletion of sequences at arbitrarily chosen positions between the 2 nick sites and can also insert a sequence, replacing the endogenous sequence with the Bxb1 attB sequence. The gene of interest with attP flanked sequence at 5′ and 3′ specifically inserted to the target site has an attB site by Bxb1…

Figure 2. Nuclease‐based approach for the deletion and translocation of genes. A) PEDAR: The Cas9 nuclease generates a double‐strand break followed by 2 3′ flaps created by paired pegRNAs that target the complementary DNA strands (pegF and pegR). The 2 3′ flaps generated by these pegRNAs are complementary. The sequence deleted in between the flap with excised by the DNA repair mechanism. The regions where DNA is excised are indicated by dark blue boxes, with the complementary 3′ flaps shown in the purple box. B) PETI: Similar to PEDAR, the Cas9 nuclease induces a double‐strand break followed by 2 3′ flaps created by paired pegRNAs that target different chromosomes and are complementary to the sequence of other target sites. This results in the translocation of chromosomes, indicated by the green and red 3′ flaps showing the regions of homology (red to red and green to green) to the targeted site. Figure created with Bio Render.com.

Figure 3. DNA polymerase‐assisted gene editing. A) SPE: This prime editing approach involves splitting the reverse transcriptase from Cas9. The reverse transcriptase (RT) and template RNA (LPET and PET) are tethered by MCP and the MS2 loop. LPET/PET anneals to the nicked DNA strand and serves as the template for RT. A single‐strand nick in the DNA by Cas9 nickase drives reverse transcription by RT, thereby incorporating the desired edit into the target site. B) DPE: In this design, RT is replaced by MCP‐tethered phi29, which binds to the DPET via MS2. The DPET anneals to the nicked DNA strand and serves as the template for phi29 polymerase (a DNA polymerase). DPET encodes the primer‐binding site and the prime‐editing template. C) CE: This system consists of a Cas9 nickase, a DNA‐dependent DNA polymerase, and a single‐stranded DNA (ssDNA) tethering domain (HUHe) paired with a guide RNA (gRNA). The clkDNA is a single‐stranded DNA oligonucleotide that encodes a PBS, a PT, and an HUHe recognition site, serving as a template for the DNA polymerase. Polymerization of the template strand and subsequent DNA repair mechanisms lead to the incorporation of the desired edit into the target strand.…

Figure 4. RT and transposable element‐based techniques for efficient integration. A) PASTE: Prime editing allows for the insertion of the attB sites in the target sequence by atgRNA (gRNA+ RT with attB site), and the gene of interest flanked by the attP sequence is inserted into the target site by the Bxb1 integrase. In the diagram, the gene of interest is shown in the violet box, the attP site in the dark blue box, and the attB site in the light blue box. B) CREATE: The CREATE system produces ORF1p and ORF2p from CREATE mRNA, which are co‐assembled into a ribonuclear complex with the CREATE mRNA. Cas9H840A and sgRNAs induce non‐target strand nicking. The ORF1p RNA‐binding protein aids in the formation of the RNP complex. The DNA 3′‐flap hybridizes with PBS1 to prime the first strand of cDNA synthesis mediated by the ORF2p RT domain, while the second strand synthesis is primed by PBS2, which hybridizes with the reverse‐transcribed PBS2. In the diagram, the PBS1 binding site is shown in the blue box, and the PBS2 binding site in the brown box. Figure created with Bio Render.com.

Figure 5. Circular RNA‐mediated gene editing for stability. The CPE (circular prime editing): A and C) Tethered RT and Cas9: In this system, RT and Cas9 are tethered together. The circular RNA encodes the primer‐binding site (PB, shown in blue box) and the reverse transcription template (RT, shown in black box) tethered to Cas9 via MCP‐MS2 interaction. B and D) Untethered RT and Cas9: here RT and Cas9 are not tethered together. The circular RNA encodes the PB (blue box) and RT (black box), with the RT interacting via MCP‐MS2. (A and B) Cas9 nuclease generates a double‐strand break at the target site, with the reverse transcriptase performing reverse transcription to incorporate the desired edits (pink box). (C and D) Cas9 nickase generates a single‐strand nick at the target site, with reverse transcription performed by the reverse transcriptase to incorporate the desired edits (pink box). Figure created with Bio Render.com.

Figure 6. Targeted RNA‐templated mediated gene Editing. Cleavage by TevSaCas9/rep‐gRNA facilitates the recruitment of Rad52, which can bind to RNA or DNA and mediate RNA strand exchange at the Tev cleavage site. This interaction leads to a hybrid RNA structure with a 3′‐OH end, which recruits DNA polymerase θ (Polθ). The repair intermediate is then resolved through a fill‐in‐gap repair process involving DNA ligase and an unidentified DNA polymerase, resulting in the completion of the repair. In the diagram, Rep RNA is shown in the black box, the gene of interest in the pink box, and the DNA flap in the grey box. Figure created with Bio Render.com.