Prime editing for precise and highly versatile genome manipulation

Abstract

Programmable gene-editing tools have transformed the life sciences and have shown potential for the treatment of genetic disease. Among the CRISPR-Cas technologies that can currently make targeted DNA changes in mammalian cells, prime editors offer an unusual combination of versatility, specificity and precision. Prime editors do not require double-strand DNA breaks and can make virtually any substitution, small insertion and small deletion within the DNA of living cells. Prime editing minimally requires a programmable nickase fused to a polymerase enzyme, and an extended guide RNA that both specifies the target site and templates the desired genome edit. In this Review, we summarize prime editing strategies to generate programmed genomic changes, highlight their limitations and recent developments that circumvent some of these bottlenecks, and discuss applications and future directions.

Figure 1 |. Precision genome editing in mammalian cells.

a | Cas nucleases can induce target DNA disrupt via formation of small insertions or deletions (indels), or DNA integration typically accompanied by substantial frequencies of undesired indel byproducts. b | Base editing can mediate C•G-to-T•A, C•G-to-G•C, and A•T-to-G•C conversions with few indel outcomes. Base editors canonically use a Cas9 nickase that only cuts the complementary strand. c | Prime editing can program any type of precise nucleotide substitutions, as well as insertions or deletions of up to hundreds of bases. Prime editors canonically use a Cas9 nickase that only cuts the non-complementary strand. Red DNA represents precisely edited sequence, and black DNA represents undesired outcomes. Blue DNA bases show the position of the protospacer-adjacent motif (PAM) required for Cas9 targeting. RuvC and HNH represent nuclease domains of Cas9. HDR, homology-directed repair; HITI, homology-independent targeted integration

Figure 2 |. Original prime editing systems.

Prime editor 1 (PE1) editing systems use a fusion of Streptococcus pyogenes Cas9 (SpCas9) nickase and Moloney murine leukaemia virus reverse transcriptase (MMLV RT) in complex with a prime editing guide RNA (pegRNA) to nick the non-complementary genomic strand and template the synthesis of an edited DNA flap. The PE2 prime editor uses an engineered MMLV RT with improved efficiency and stability. The edited 3ʹ flap is processed by endogenous cellular pathways to permanently copy the edited sequence to the non-edited strand. PE3 editing systems use an additional single-guide RNA (sgRNA) to direct the PE2 enzyme to nick the non-edited strand and stimulate replacement of the non-edited strand, which enhances permanent incorporation of the edited sequence.

Figure 3 |. Advancements in prime editing systems.

a | Variants of prime editors or prime editing guide RNAs (pegRNAs) that ultimately enhance the creation of the edited 3ʹ DNA flap. PEmax, IN-PE2, pegRNAs carrying the F+E crRNA scaffold^,, and ePE pegRNAs improve prime editor and pegRNA expression. epegRNAs, ePE pegRNAs, xr-pegRNAs, G-PE pegRNAs, and sPE pegRNAs reduce pegRNA degradation. PE2*, PEmax, CMP-PE-V1⁶³, and hyPE2 improve localization and/or DNA targeting of the prime editor complex. PEmax improves nicking of the genomic DNA strand. ePPE assists pegRNA-primer annealing. The engineered MMLV RT in PE2 strongly enhances DNA flap synthesis. b | Strategies that promote permanent incorporation of the edited 3ʹ flap into genomic DNA. DNA ligation of the 3ʹ nicked heteroduplex intermediate followed by DNA replication successfully incorporates the desired prime edit, but mismatch repair of this intermediate excises and replaces the edited strand, resulting in no prime editing. Nicking the non-edited DNA strand with PE3 promotes copying of the edit to both genomic strands. Cellular mismatch repair excises the nicked strand of DNA heteroduplexes, commonly leading to removal of the edit. Transient inhibition of mismatch repair with MLH1dn (PE4 and PE5) or with small interfering RNAs (siRNAs)^,, or mismatch repair evasion through judicious design of the 3ʹ flap, enhances conversion to the desired prime editing product and reduces undesired formation of small insertions or deletions (indels).

Figure 4 |. Prime editing variants.

a | Prime editing with paired prime editing guide RNAs (pegRNAs) that template two 3ʹ DNA flaps containing the edit^–,,–. DNA intermediates formed after prime editor (PE)-mediated nicking and flap extension at the pegRNA target sites are shown on the left. Annealing of the complementary flaps, excision of the original genomic duplex, and ligation of the resulting nicks can efficiently mediate small edits or large insertions and deletions. b | Prime editing with serine integrases enables the targeted insertion of gene-sized (>1 kilobase) DNA segments. Twin prime editing or PASTE first installs an attB site into the genome using prime editing, then uses Bxb1 recombinase to mediate integration of donor DNA into the site^,. c | Prime editors containing Cas9 nuclease create double-strand breaks (DSBs) with an edited 3ʹ overhang that can be incorporated into the genome but also result in frequent formation of small insertion or deletion (indel) byproducts^,,. Red DNA bases represent edited DNA with heterologous sequence. Blue and orange DNA bases represent homologous sequence. ∆ specifies DNA bases deleted by the edit. Scissors denote sites of Cas9-induced DSBs.