Development and Application of Prime Editing in Plants

doi:10.1016/j.rsci.2023.07.005

Abstract

Abstract:

Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-mediated genome editing has greatly accelerated progress in plant genetic research and agricultural breeding by enabling targeted genomic modifications. Moreover, the prime editing system, derived from the CRISPR/Cas system, has opened the door for even more precise genome editing. Prime editing has the capability to facilitate all 12 types of base-to-base conversions, as well as desired insertions or deletions of fragments, without inducing double-strand breaks and requiring donor DNA templet. In a short time, prime editing has been rapidly verified as functional in various plants, and can be used in plant genome functional analysis as well as precision breeding of crops. In this review, we summarize the emergence and development of prime editing, highlight recent advances in improving its efficiency in plants, introduce the current applications of prime editing in plants, and look forward to future prospects for utilizing prime editing in genetic improvement and precision molecular breeding.

Key words: prime editing, CRISPR/Cas, precision genome editing, crop breeding

Liu Tingting, Zou Jinpeng, Yang Xi, Wang Kejian, Rao Yuchun, Wang Chun. Development and Application of Prime Editing in Plants[J]. Rice Science, 2023, 30(6): 509-522.

Figures/Tables 4

Fig. 1. Composition and mechanism of prime editing (PE) system. A, Composition of original PE system. The PE system is composed of two components: prime editor and pegRNA. The prime editor is a fusion protein formed by nCas9 and wild type MMLV reverse transcriptase. The pegRNA is an engineered sgRNA with an added PBS for directing prime editor to the genomic target site and a RTT for mediating the desired edit at the 3?-terminus of sgRNA. MMLV, Moloney murine leukaemia virus reverse transcriptase; nCas9 (H840A), Cas9 nickase; nCas9-MMLV, A fusion protein formed by nCas9 and MMLV; pegRNA, Prime editing guide RNA; PBS, Primer binding site; RTT, Reverse transcriptase template; sgRNA, Single-guide RNA. B, Mechanism of PE system. PE system uses a prime editor with a pegRNA to nick the PAM-containing strand and template the synthesis of an edited DNA flap. The resulting 3?-end hybridizes to the PBS, then primes reverse transcription of new DNA containing the desired edit using the RTT of the pegRNA. PAM, Protospacer adjacent motif. C, Overview of DNA repair process. The edited 3?-flap is processed by endogenous cellular pathways, and after initial synthesis of the edited strand, the 5?-flap is excised leaving a DNA heteroduplex containing one edited strand and one non-edited strand. Mismatch repair resolves the heteroduplex by permanently copying the edited sequence to the non-edited strand.

Fig. 2. Emergence and development of prime editing (PE) systems in plants. A, Emergence of plant PE (PPE) systems. PE2 prime editor uses an engineered MMLV with improved efficiency and stability. Building upon PE2 system, PE3 system uses an additional sgRNA to nick the non-edited strand and stimulate replacement of the non-edited strand, which enhances permanent incorporation of the edited sequence. The sgRNA of PE3b matches the edited strand, mismatches the unedited allele, and only cleaves and repairs after the edited strand has been edited. MMLV, Moloney murine leukaemia virus reverse transcriptase; PBS, Primer binding site; RTT, Reverse transcriptase template; sgRNA, Single-guide RNA. B, Development of PE in various plants. PEs were performed in rice, wheat, maize, and tomato with endogenous gene editing.

Fig. 3. Overview of prime editor (PE) system optimization in plants. A, Various types of prime editors. PPE1 is the original PE system, and PPE2 is updated to engineering MMLV. On the basis of PPE2, PPEmax is changed to nCas9 variant and a c-Myc NLS is added. ePPE is updated to MMLV-△RHase H and an NC protein is added between nCas9 and MMLV. PPEmax-MLH1dn has one more individually expressed MLH1dn protein than PPEmax. PPE-T5 adds a T5 exonuclease prior to nCas9. PrimeRoot adds an integrase (Cre/FLP) and a nucleoplasmin NLS than ePPE at the end. PPE-SpG is updated to nSpG. NC, A viral protein with nucleic acid chaperone activity that affects a variety of functions related to reverse transcription; NLS, Nuclear localization signal; PPE, Plant prime editing system; Pro, Promoter; nCas9, Cas9 nickase; MMLV, Moloney murine leukaemia virus reverse transcriptase; MMLV-△RHase H, A MMLV variant with deletion ribonuclease H (RNase H) domain; MLH1dn, A dominant negative variant of MLH1 protein; nSpG, An nCas9 variant with NGN (N = A, T, C, G) protospacer adjacent motif. B, Various types of pegRNA. The original pegRNAs are optimized for complex promoter-driven or motif-added pegRNAs to improve PPE efficiency. OsU3, Rice U3 promoter; CaMV35S-CmYLCV-U6, Composite promoter of the CaMV 35S enhancer, CmYLCV promoter, and shortened U6 promoter; evopreQ1, A structured RNA pseudoknot that protects 3?-extension from degradation by exonucleases; MS2, A hairpin that facilitates its recruitment to reverse transcriptase by binding a fused MS2 coat protein. C, Various types of twinPE strategies. The dual-pegRNA and GRAND mediated precise insertion or deletion of DNA fragments. The edited or insertion (or deletion) is labeled in red. PBS, Primer binding site; RTT, Reverse transcriptase template with edit; pegRNA, Prime editing guide RNA; GRAND, An editing strategy, genome editing by RTTs partially aligned to each other but nonhomologous to target sequences within dual-pegRNA.

Fig. 4. Applications of prime editing systems in plants. A, Creation of herbicide-resistant germplasms. The EPSPS, ACC1, and ALS genes are mainly modified to enhance herbicide resistance. B, Creation of effective alleles. The saturation mutation of P1927 in the OsACC1 yields three mutation types, which are indicated by red horizontal lines in the figure. P, Proline; Y, Tyrosine; F, Phenylalanine. C, Insertion of protein tags. Insert different tags at the N-terminus (N-tag) and C-terminus (C-tag), such as 6× His, HA and FLAG. D, Regulation of protein expression. Down-regulation of downstream target protein expression by expanding endogenous uORFs (Type A) and generating de novo uORFs (Type B) strategies. WT, Wild type; UTR, Untranslated region; 6× His, Polyhistidine tag; HA, Hemagglutinin tag; uORF, Upstream open reading frame; pORF, Primary open reading frame.

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