TABLE 2.
Strategies used for optimization of the PE system.
| Strategies | Description | Results | References |
|---|---|---|---|
| Optimization of pegRNA | |||
| PRIME-Del | The technology is used for accurately editing the targets based on a pair of pegRNAs for opposite DNA strands | Targeted deletion of sequences up to 10 KB with higher accuracy (1%–30%). | Choi et al. (2022) |
| GRAND | The technology is based on a pair of pegRNAs with different but complementary RTT and target DNA sequences | Its editing efficiency was 63.0% in 150 bp inserts but it could introduce a small amount of by-products, and its editing efficiency was reduced to 28.4% in 250 bp insertions | Wang et al. (2022) |
| Bi-PE | The Bi-PE system contains nick sgRNA near the pegRNA template sequence | The Bi-PE strategy can increase efficiency by 16 times and the editing accuracy by 60 times. The system can delete large DNA (100–100 bp) and can introduce small fragments of 10–100 bp into the deletion sites | Tao et al. (2022b) |
| HOPE | Two pegRNAs which contain homologous 3′terminals and used for targeting DNA double strands are designed in this system | HOPE provides a new choice of guided editing with a high balance between efficiency and accuracy | Zhuang et al. (2022) |
| twinPE | The system uses a prime editor protein and two pegRNAs | The length of genes that can be edited by twinPE has expanded to thousands of base pairs which are equivalent to the length of a complete gene | Anzalone et al. (2022) |
| ePE | The technology is based on the optimization of the pegRNA skeleton through deletion and replacement | Compared with the standard PE, ePE improves the editing efficiency of point mutation by an average of 1.9 times | Liu Y et al. (2021b) |
| epegRNAs | The engineered pegRNA (epegRNAs) based on integration of the structured RNA motifs into the 3′end of pegRNAs | The optimized PE system can increase the editing efficiency in HeLa, U2OS, K562 cells and primary human fibroblasts by 3–4 times without increasing the off-targeted editing activity. | Nelson et al. (2022) |
| xrPE | The xrPE platform is developed by adding an xrRNA to the 3′extension region of pegRNAs | The average enhancement in base transformation, small deletion and small insertion of pantarget was 3.1, 4.5, and 2.5 times, and xrPE has comparable edits/index ratios, and minimum deviation target edits | Zhang et al. (2022) |
| TM of PBS | Optimization of PBS and RT template length | When the TM of PBS was at about 30°C, the activity of PE was 1.5–4.3 times higher than that of the PBS with other TM | Lin et al. (2021) |
| Length of RT template | The ratio of targeted editing to off-targeted editing was significantly affected by the length of RT template, but did not change with the length of PBS or the location of nicking sgRNA | Lin et al. (2020) | |
| apegRNA and spegRNA | The apegRNA is developed by improving the pegRNA secondary structure and the spegRNA is developed by introducing same-sense mutations (SSM) at proper positions | The frequency of indel increased, but the frequency of unexpected indel and the part of incomplete products and by-products were not significantly affected | Li et al. (2022). |
| Optimization of the effecter proteins | |||
| PE2 variants | PE2 variants (PE2-vqr, PE2-vrqr, PE2-vrer, PE2-ng, PE2-spg, and PE2-spry) recognize the different PAM sequences | More than 50 types of PE2 variants were successfully generated in HEK293T cells, and their editing activity was as high as 51.7%. In addition, BRAF V600E mutation was successfully introduced, which could not be induced by traditional PE system | Kweon et al. (2021) |
| ePPE | The ePPE system is constructed by deleting the RNase H domain in M-MLV RT and adding virus nucleocapsid protein (NC) | The synergistic effect of the two modifications increased the efficiency of base substitution, deletion and insertion at different endogenous sites by an average of 5.8 times, while no significant increase in by-products or partial off-targeted editing was observed | Zong et al. (2022). |
| WT-PE | The WT-PE system is designed by fusing reverse transcriptase (RT) and nuclease wild-type Cas9 | WT-PE has realized the efficient and multifunctional large-scale genome editing, including large-scale deletion of up to 16.8 Mbp and chromosome translocation. This system could generate bi-directional primer editing | Tao et al. (2022a) |
| FnCas9 | A technique developed by connecting reverse transcriptase to the new Francisella novicida Cas9 (FnCas9) | The editable region of prime editing could be extended by different nicking properties of CRISPR-Cas orthologs and engineering the PAM recognition domain within the Cas9 nickase. It also expands the region recognized as RTT after PBS sequence for prime editing | Oh et al. (2022). |
| PepSEq | A technique developed by fusing peptides derived from DNA repair proteins to the N-terminal of PE2 | Through peptide fusion, PepSEq significantly improved the prime editing efficiency | Velimirovic et al. (2022) |
| Split-PE | Fusion proteins are split into two parts: split nSpCas9 and MMLV-RT | This PE system can be tested faster without optimizing the length or relative position of the linker with the fusion protein. In addition, off-target effects of split PE2 is similar to that of PE2 | Grunewald et al. (2022) |
| Collaborative optimization of PE with multiple strategies | |||
| PEDAR | PEDAR is based on the Cas9 nuclease (but not nCas9) combined with the reverse transcriptase and a pair of pegRNA | PEDAR can introduce more than 10 kb target deletion and up to 60 bp insertion into cells. In the tyrosinemia mouse model, PEDAR removed the 1.38-kb pathogenic insertion in the Fah gene and accurately repaired the missing connection to restore the expression of the Fah gene in the liver | Jiang T et al. (2022a). |
| The optimized PE | In the optimized PE system, RT is fused at the N-terminal of nCas9 and multiple nucleotide substitutions are introduced into the reverse transcriptase template | The optimized PE system was applied in the transgenic rice plants (24.3%), the maize protoplasts (6.2%) and the human cells (12.5%), which was 2–3 times higher than PE3 | Xu et al. (2022) |
| SPE | The pegRNAs are split into sgRNA and petRNA, replace M-MLV RT with two compact codon optimized bacterial RT, use a dual AAV vector strategy | The efficiency in installing accurate editing is similar to that of PE3 (30%), while indel by-products are not increased, and AAV delivery is simpler | Liu et al. (2022) |
| Optimization strategy based on inhibiting DNA mismatch repair | |||
| PE4, PE5 and PEmax | The PE4 and PE5 systems are constructed through transient expressing the DNA mismatch inhibitor protein MLH1dn coding gene. PEmax is constructed by changing RT codon, mutating SpCas9 and optimizing the NLS sequence | The editing efficiencies of PE4 and PE5 systems are 7.7 times and 2.0 times higher than that of PE2 and PE3 respectively, and the editing ratio is 3.4 times higher | Chen et al. (2021) |
| Comprehensive optimization method including three strategies | |||
| ePE3max and ePE5max | ePE3max consists of PEmax protein, an epegRNA with evopreQ1 and a nicking sgRNA, and ePE5max consists of the ePE3max system and a dominant negative OsMLH1 variant that inhibits MMR | The prime editing efficiency of rice is greatly increased by improving PEmax structure and epegRNA. In addition, the by-products derived from pegRNA scaffold can be eliminated by using the termination rule of designed pegRNA | Jiang et al. (2022) |