Development and Application of a High-Efficiency Dual-Base Editing Tool for Solanaceous Crops

doi:10.7668/hbnxb.20195725

Abstract

Abstract:

This study seeks to develop a highly efficient dual-base editing tool for solanaceous crops to address the challenges of poor compatibility and low editing efficiency associated with existing base editing systems in these plants. The research involved fusing the nickase Cas9 enzyme (nCas9),which has partially lost its nuclease function,with components such as the highly efficient adenine deaminase ABE8e,the optimized cytosine deaminase sdd7-mini,and the uracil glycosylation inhibitor UGI. Systematic optimization was conducted based on the promoter preferences and codon usage frequencies specific to solanaceous crops. Ultimately,the dual-base editor TPDBE-S was constructed. In transient transformation experiments using tomato and eggplant protoplasts,this editor exhibited exceptionally high editing efficiency. In the stable transformation system of tobacco,targeting the PDS gene verification revealed that four out of five positive transgenic families exhibited pronounced albinism phenotypes,thereby confirming the stable editing capability. The editing window of this editor aligns closely with the characteristics of ABE8e and sdd7-mini. Furthermore,optimizing codon preference and promoter activity in Solanaceae crops significantly enhances editing efficiency and adaptability. The construction of TPDBE-S employs a modular design. The multi-target expression cassette,based on the tRNA self-cleaving mechanism,can be rapidly assembled through one-step gene synthesis,which is both cost-effective and straightforward. This tool addresses the gap in double-base editing specifically for Solanaceous crops.

Key words: Eggplant, Gene editing, Dual-base editing, Tomato, Editing efficiency

CLC Number:

TIAN Peng, LI Yadong, ZHANG Jingjing, TIAN Zhejuan, KANG Chen, GAO Xiurui, LI Bing, LIU Wei, WU Yanrong, WU Zhiming. Development and Application of a High-Efficiency Dual-Base Editing Tool for Solanaceous Crops[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 39-48. doi: 10.7668/hbnxb.20195725.

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URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20195725

http://www.hbnxb.net/EN/Y2026/V41/I1/39

Figures/Tables 7

Tab.1 List of PCR primers

引物名称 Name	引物序列(5'—3') Primer sequence(5'—3')	用途 Purpose
T-A-180F	CCTCCAAACCCTCCACCCCA	检测编辑位点T-A
T-A+400R	CTCAGGCATAGCTGTTTCCTGCC	检测编辑位点T-A
T-B-60F	CGGAGGGGAGAGCTGAAATGGCT	检测编辑位点T-B
T-B+500R	AACCAGTCAGTCCACTGCTCCA	检测编辑位点T-B
T-C-150F	TGGGACGGGCGGAGATGGTT	检测编辑位点T-C
T-C+200R	CACCGCTCGAACCTGTCACAT	检测编辑位点T-C
E-2350F	TGCGTGTCGGATTCGCCAAAA	检测编辑位点E-A/B/C/D
E-4350R	CCTGCGAAAGGGCTTCCTCTGC	检测编辑位点E-A/B/C/D
Cas9-440F	TCCTCTAGCAAGTGGACCAACGTA	检测烟草转基因阳性植株
Cas9-440R	ACAAAGGCTCCTCTTTCAGCTTCTA	检测烟草转基因阳性植株

Fig.1 Schematic diagram of the double-base editor TPDBE-S structure 35S.Cauliflower mosaic virus 35S RNA promoter;NLS.Nuclear localization signal;Sdd7-mini.Cytosine deaminase;TadA8e.Adenine deaminase;32aa/39aa.Linker peptides of lengths 32aa/39aa;DBD.DNA-binding domain;nCas9.Cas9 nickase with partially lost nuclease function;UGI.Uracil glycosylase inhibitor;P2A.Self-cleaving sequence derived from porcine teschovirus;GFP.Green fluorescent protein;Tnos.Transcription terminator;SiU6.Tomato SiU6 RNase Ⅲ promoter;tRNA.Transfer RNA sequence(77 bp);gRNA.Guide RNA sequence(96 bp);Poly T.Transcription termination signal;HygR.Hygromycin phosphotransferase;LB/RB.Left and right borders for binary vector insertion into the plant genome.The red dashed box.Fragment that needs to be synthesized when editing the target genes.

Fig.2 Schematic diagram of the double-base editor TPDBE-S structure and enzyme digestion verification A.Schematic diagram of the double-base editor TPDBE-S structure.This diagram was drawn using SnapGene V6.0.2 software,with key restriction sites highlighted.B.Enzyme digestion verification of the double-base editor TPDBE-S: M.Marker,1.TPDBE-S plasmid,2.TPDBE-S Plasmid Spe Ⅰ digestion,3.TPDBE-S plasmid Sma Ⅰ digestion,4.TPDBE-S plasmid BamH Ⅰ digestion.

Fig.3 Schematic diagram of the double-base editor TPDBE-S gRNA expression cassette sequence and structure The underlined portions at the start and end of the sequences.The homologous arm sequences flanking the Spe Ⅰ restriction site on the TPDBE-S vector,Red font.The Spe Ⅰ restriction site;Blue shaded portions.The tRNA sequences;Yellow shaded portion.The tracrRNA sequences;T1—T3.CRISPR-Cas9-targeted sequences;The gray shaded portion.The Poly T termination signal.

Tab.2 Statistics on editing efficiency at different sites

位点 Site	物种 Species	基因号 Gene number	靶标序列 Target sequence	总编辑效率 Overall editing efficiency	A-G的编辑效率 Editing efficiency of A-G	C-T的编辑效率 Editing efficiency of C-T	A-G&C-T的编辑效率 Editing efficiency of A-G&C-T
T-A	番茄	Solyc01g105270	TCAAAGATGGGGCGAACTCG TGG	86.7%(26/30)	66.7%(20/30)	60.0%(18/30)	40.0%(12/30)
T-B	番茄	Solyc02g080370	ACGTCAACGAATCCGTTCAG TGG	80.0%(24/30)	53.3%(16/30)	60.0%(18/30)	33.3%(10/30)
T-C	番茄	Solyc01g007900	CAACGCCGTAGAGAACAGCA AGG	53.3%(16/30)	0(0/30)	53.3%(16/30)	0(0/30)
E-A	茄子	Smechr0602754	ACTTCAAAGGGCTAGCTCTC AGG	66.7%(20/30)	36.7%(11/30)	46.7%(14/30)	16.7%(5/30)
E-B	茄子	Smechr0602754	TGTTTGACATGGAATATTCA AGG	55.3%(16/30)	53.3%(16/30)	3.3%(1/30)	3.3%(1/30)
E-C	茄子	Smechr0602754	TTGTAATGCAGTTCGAAGCT CGG	90.0%(27/30)	70.0%(21/30)	56.7%(17/30)	36.7%(11/30)
E-D	茄子	Smechr0602754	GAACGCTGCTTCCTTTGGAT GGG	63.3%(19/30)	0(0/30)	63.3%(19/30)	0(0/30)

Fig.4 Editing efficiency of the dual-base editor TPDBE-S in the stable transformation system of Nicotiana tabacum A.Phenotypes of positive transgenic plants(lines #1—#5)obtained via Agrobacterium-mediated transformation with TPDBE-S.The albinotic sectors,indicative of targeted mutagenesis,are indicated by red arrows.Scale bar.1 cm.B.PCR-based genotyping of transgenic N.tabacum T-DNA insertion events: M.DNA molecular-weight Marker,CK+.Positive control(TPDBE-S plasmid),CK-.Negative control(H₂O),#1—#5.PCR products corresponding to the five independent transgenic lines.

Fig.5 Overview of editing window and frequency of TPDBE-S at various target sites Blue characters.PAM sites;Red characters.The edited sites.

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