Prime editing: Mechanism insight and recent applications in plants

Abstract

Prime editing (PE) technology utilizes an extended prime editing guide RNA (pegRNA) to direct a fusion peptide consisting of nCas9 (H840) and reverse transcriptase (RT) to a specific location in the genome. This enables the installation of base changes at the targeted site using the extended portion of the pegRNA through RT activity. The resulting product of the RT reaction forms a 3' flap, which can be incorporated into the genomic site through a series of biochemical steps involving DNA repair and synthesis pathways. PE has demonstrated its effectiveness in achieving almost all forms of precise gene editing, such as base conversions (all types), DNA sequence insertions and deletions, chromosomal translocation and inversion and long DNA sequence insertion at safe harbour sites within the genome. In plant science, PE could serve as a groundbreaking tool for precise gene editing, allowing the creation of desired alleles to improve crop varieties. Nevertheless, its application has encountered limitations due to efficiency constraints, particularly in dicotyledonous plants. In this review, we discuss the step-by-step mechanism of PE, shedding light on the critical aspects of each step while suggesting possible solutions to enhance its efficiency. Additionally, we present an overview of recent advancements and future perspectives in PE research specifically focused on plants, examining the key technical considerations of its applications.

Keywords: CRISPR-Cas; precise gene editing; precision plant breeding; prime editing; synthetic biology.

Figure 1

Overview of the PE mechanism. (a) Schematic diagram illustrating the components involved in the PE mechanism. The PE system comprises three main components: (1) nCas9 (H840A) proteins (faint blue colour), which induce single‐strand cleavage on the non‐target strand (light purple strand); (2) M‐MLV RT (pink colour), fused to nCas9 (H840A) via amino acid linkers, responsible for synthesizing a new single‐strand DNA incorporating desired edits (red coloured sequences); (3) pegRNA, containing a primer binding site (PBS) and an RT template that extends the 3′ end of a single‐guide RNA (sgRNA). The PE complex is guided to complementary sequences (target sequences) in the pegRNA spacer sequence. nCas9 (H840A) cleaves the nucleotide at the −3 position from the PAM site on the non‐target strand. The PBS (green‐coloured sequences) at the 3′ end of pegRNA binds to the complementary sequence on the non‐target strand, resembling a ‘primer’. M‐MLV RT initiates the synthesis of a new single‐strand DNA using the exposed 3′ hydroxyl group of the ‘primer’ and the RT template as a template. After synthesis, the newly synthesized single‐strand DNA competes with the original strand for insertion into the genome. If the original strand is preferentially installed in a 5′ flap while the new strand is removed, the existing genome sequence remains unchanged. Conversely, in a 3′ flap preference scenario, the new strand is preferentially installed while the original strand is removed, enabling desired edits to be introduced. Successful binding of the 3′ flap activates a mechanism to repair mismatched edit sequences. This can result in restoring the wild‐type (WT) sequence through the MMR pathway or the complete installation of the edit sequence by repairing the target strand. Additional cleavage of the target strand can trigger the installation of the edited strand. Green sequence: PBS; Red sequence: edit sequence; Faint blue protein: nCas9 (H840A); Pink protein: M‐MLV RT; Light purple strand: non‐target strand; Navy strand: target strand; Orange: spacer and scaffold of pegRNA. (b) Components of the PE system and various approaches that have enhanced prime editing efficiency.

Figure 2

PE components and enhanced features. (a) Schematic representation of an approach involving 3′ extension protection by pseudoknots. Engineered pegRNA incorporates a pseudoknot structure at its 3′ end to safeguard the 3′ extension from exonuclease degradation. The secondary structures of several pseudoknot variants tested for enhancing prime editing efficiency are depicted. evopreQ1: modified prequeosine1‐1 riboswitch aptamer; tevopreQ1: evopreQ1 with a trimmed (grey at 5′ end) sequence; mpknot: frameshifting pseudoknot from M‐MLV; tmpknot: mpknot with a trimmed (grey at 3′ end) sequence; xrn1: Xrn1‐resistant RNA from sweet clover necrotic mosaic dianthovirus (SCNMV). (b) Protein‐based engineering strategies for PE. PE2: The original M‐MLV RT of PE1 is modified to enhance thermostability and DNA–RNA substrate affinity; PE2max: Contains additional modifications in nCas9 (H840A) to improve Cas9 nuclease activity. ePPE: Engineered Plant Prime Editor, where the NC is fused between nCas9 (H840A) for nucleic acid chaperone activity related to reverse transcription, and the ∆RNase H domain of M‐MLV is deleted to inhibit RNase H‐directed degradation of RNA–DNA heteroduplex. sPE: Separated expression of nCas9 (H840A) and M‐MLV RT for adeno‐associated viral (AAV) vector packaging capacity; petRNA: Split pegRNA into sgRNA and RT template‐PBS. The RT template‐PBS sequence is engineered into a circular form with the addition of the MS2 aptamer; sPE with petRNA: sgRNA guides the nCas9 (H840A)‐RT effector to the target site, and circularized petRNA is tethered to the MCP‐RT fusion protein by MS2. The PBS sequence of the tethered petRNA binds to a complementary sequence, and M‐MLV RT transcribes new single‐strand DNA using the RT template sequence. PE4: PE and MLH1dn are expressed independently. MLH1dn inhibits the MMR pathway, thereby increasing the probability of repair by the target strand. (c) pegRNA‐based strategies. Paired pegRNAs: The 3′ flap of two pegRNAs shares partial or complete complementary sequences. apegRNA‐2: the scaffold sequence of pegRNA is altered to stabilize the secondary structure by replacing a non‐C/G pair with a C/G pair in the small hairpin. RNA Pol III promoter: The Pol III promoter is combined with the widely used U6 promoter for sgRNA expression to enhance the transcription level of pegRNA. spegRNA: Introduction of synonymous mutations before or after the edit sequence to increase the efficiency of PE.

Figure 3

PE approach using paired pegRNAs and major variants. (a) The PE approach utilizes two pegRNAs for targeted editing. Here, we illustrate the target sequence and pegRNAs designed to introduce an herbicide‐resistant allele (P186S: CCA to tCt) into the tomato ALS1 locus. The paired pegRNAs consist of a forward pegRNA (pegRf) and a reverse pegRNA (pegRr), enabling PE on both strands in close proximity. The RT templates are designed to fully complement each other, facilitating annealing and accurate incorporation of the desired base changes (represented by the discontinuous blue box) into the genomic site. (b) Variants of paired pegRNAs. The paired PE variants differ in selecting complementary 3′ extensions, which can be entirely homologous (indicated with the same colour) to the genomic sequence except for the desired base changes, allowing for precise editing in paired PE. Other variants may involve the omission of genomic fragments (PRIME‐Del/bi‐PE) or the omission of genomic fragments while introducing new sequences in the edits (twinPE/PRIME‐Del/bi‐PE/GRAND). In specific cases requiring long‐range DNA fragment deletion, fully functional SpCas9 is utilized for more efficient PE (C1‐WT‐PE and C2‐WT‐PE).

Figure 4

SSR‐mediated precise DNA insertion approaches. (a) Overview of the recently reported SSR‐mediated precise DNA insertion approaches. (b) TwinPE and PASTE‐based DNA insertion using serine SSRs. The TwinPE approach inserted the attP site into the genomic site and placed the attB site within the donor template for recombination. Reversely, the PASTE approach first inserted the attB into the genomic site, and the attP site was used with the donor template for integration. Bxb1 integrases catalyse sequence exchange between the recombination sites (attB with attP or attP with attB), resulting in the insertion of the donor DNA template into the targeted site. (c) Steps involved in PrimeRoot‐mediated DNA insertion using tyrosine SSRs in plants. The PrimeRoot approach utilizes Cre recombinase, a tyrosine recombinase, to mediate DNA insertion at selected safe‐harbour sites within the plant's genomes. Paired pegRNAs are used to instal the lox66 sequence at the targeted site. A circularized double‐stranded DNA (dsDNA) donor containing the donor DNA template and a lox71 sequence is introduced. Site‐specific recombination between lox66 and lox71 mediated by Cre recombinases, generating nicks (indicated as scissors), results in the precise insertion of the DNA carried by the circularized dsDNA donor.