Yuming Lu, Yifu Tian, Rundong Shen, Qi Yao, Dating Zhong, Xuening Zhang, Jian-Kang Zhu

Plant Biotechnol J. 2021 Mar; 19(3):415-417.  doi: 10.1111/pbi.13497. Epub 2020 Nov 25.

The CRISPR/Cas‐mediated genome editing technology has been widely applied to create knockout alleles of genes by generating short insertions or deletions (indel) in various plant species. Due to the low efficiency of homology‐directed repair (HDR) and difficulties in the delivery of DNA template for HDR, precise genome editing remains challenging in plants. A tandem repeat‐HDR method was developed very recently for sequence replacement in rice, which is most useful for monocots. Base editors developed from Cas9 nickase fusion with cytosine and adenine deaminases enable targeted C‐to‐T or A‐to‐G substitutions, but are restricted to specific types of base replacements and target site selections. A ‘search‐and‐replace’ method, also known as prime editing, was developed in mammalian cells, which enables user‐defined sequence changes on a target site without requiring DSBs or the delivery of DNA repair templates. Several research groups have adopted this method for use in monocotyledonous plants, including rice and wheat. For reasons that are still unclear, although base editing has been highly efficient in monocots such as rice, its efficiencies are very low in dicots. Whether prime editing can be used for dicotyledonous plants such as tomato, is unknown. Here, we report successful adoption of prime editors for use in tomato through codon and promoter optimization. The prime editing system consists of three parts: an nCas9‐MMLV (engineered Moloney murine leukaemia virus reverse transcriptase) fusion protein, a prime editing guide RNA (pegRNA) and a small guide RNA (sgRNA) for nicking. We incorporated the mammalian prime editing system into a plant binary vector for expression in tomato, generating pCXPE01. The commonly used CaMV 35S promoter (2x35S) was used to express the nCas9‐hMMLV (human codon‐optimized MMLV) fusion protein while pegRNA and sgRNA were driven by the U6 promoter of Arabidopsis. In order to test whether the system may work in tomato, we constructed a dual‐luciferase reporter system, where the NanoLuc, an engineered super sensitive luciferase, was completely disabled by introducing frame‐shift mutations, a two nucleotide deletion and six nucleotide substitution (NanoLucM). Only precise editing on NanoLucM can restore its luciferase activity, and the efficiency could be sensitively quantified through luminescence measurement, using the firefly luciferase as an internal control. Two pegRNAs, pegRNA‐12 and pegRNA‐13, were designed with 13 and 14 nt PBS (primer binding site), respectively, and a 23 nt RT (reverse transcription) template. Each was accompanied by a sgRNA for nicking at a site located 32‐nt or 49‐nt downstream from the pegRNA nicking sites. The two pCXPE01 constructs were each introduced into tomato leaves together with the Dual‐LucM reporter using biolistic bombardment. Five days later, we detected the restored luminescence in both samples of pegRNA‐12 and pegRNA‐13, with an average efficiency of 0.26% compared with the control Dual‐Luc that was counted as 100%. These results indicated that the primer editor can be used in tomato.

See: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955883/

Figure 1: Prime editing for precise genome modification in tomato. (a) Schematic diagram of the prime editing constructs in this study. (b) Dual‐luciferase reporter system for assessments of prime editing efficiencies. Dual‐LucM contains an inactive NanoLuc designated as NanoLucM. pegRNA‐12 and pegRNA‐13 target the mutated site to restore the NanoLuc activity. fLuc, firefly luciferase. (c) Comparison of prime editing efficiencies of pCXPE01, pCXPE02 and pCXPE03 in tomato using the Dual‐luciferase reporter system delivered by bombardment. Editing frequencies were calculated by NanoLuc/fLuc, counting the normal reporter Dual‐Luc as 100%. Values (mean ± s.e.m.) were calculated from three independent experiments (n = 3). P values were obtained using the two‐tailed Student’s t‐test. (d and e) Regenerated tomato shoots (d, indicated by arrows) and a representative T0 seedling (e) on hygromycin‐containing medium. Bar, 10 mm. (f) Summary of prime editing results of pCXPE03 in regenerated tomato shoots and T0 plantlets, as determined by NGS and Sanger sequencing, respectively. (g and h) Sequence chromatograms of prime‐edited T0 plants. Edited bases were indicated by red arrows. (b, f, g and h) Targets and their PAMs in sequences were underlined in black and red, respectively. PBS and RT sequences are underlined with solid and dashed lines, respectively. Nucleotides for substitutions are marked in red.