CN118185908A - A cytosine base editing system derived from adenosine deaminase - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a cytosine base editing system derived from adenosine deaminase. The invention aims to develop a CBE tool library in plants and discover a CBE editor with better editing efficiency and editing specificity. The technical scheme of the invention is that fusion protein TadCBEa with cytosine base editing function comprises TadA-CDa deaminase and Cas protein; the TadA-CDa deaminase is obtained by modifying TadA-8e with adenosine deaminase function to perform cytidine deaminase function. The invention also provides a cytosine base editing system derived from an adenosine deaminase, comprising a fusion protein TadCBEa constructed by fusing TadA-CDa deaminase and a Cas protein. TadCBEa can realize the base editing function from C to T, and is a cytosine base editing tool with high activity and high specificity.

Description

A cytosine base editing system derived from adenosine deaminase

Technical Field

The present invention belongs to the technical field of genetic engineering, and specifically relates to a cytosine base editing system derived from adenosine deaminase.

Background technique

CRISPR-Cas technology is a widely used gene editing technology that uses RNA to guide specific binding to specific target sequences on the genome and cut DNA to form double-strand breaks, and then uses biological non-homologous end joining or homologous recombination to perform site-specific gene editing.

The development of single-base editing tools enables precise modification of specific gene sites without causing double-strand breaks in DNA. The basic principle is to fuse cytidine deaminase or adenosine deaminase with Cas protein, relying on the CRISPR principle to achieve single base modification of the target. In the single-base editing system, the base editor is mainly composed of two parts: Cas protein and DNA modification enzyme. Currently, there are two main types of base editors: cytosine base editor (CBE) based on cytidine deaminase, which realizes the conversion from C to T; adenine base editor (ABE) based on adenosine deaminase, which realizes the conversion from A to G.

The CBE systems that have been reported include: rAPOBEC1, hAID, PmCDA1, hAPOBEC3A and hAPOBEC3B. The combination of cytidine deaminase and Cas protein can achieve base editing from C to T. On this basis, the addition of one or more uracil DNA glycosylase inhibitors (UGI) can further improve the editing efficiency and product purity of base editing from C to T. The cytosine base editing systems that have been discovered can already perform effective and precise editing at specific sites, but studies have shown that rAPOBEC1-CBE has detected genome-wide off-target effects in mouse embryos and rice plants (Zuo et al., Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. 2019, Science) (Jin et al., Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. 2019, Science). Researchers are committed to finding cytosine base editing systems with higher editing efficiency, better editing specificity and better universality.

The adenosine deaminases in the ABE system include TadA-7.10, TadA-8e and TadA-9, which can perform efficient A to G base editing in human cells and plants.

Summary of the invention

The purpose of the present invention is to expand the CBE tool library in plants and discover CBE editors with better editing efficiency and editing specificity.

The present invention provides a TadA-CDa deaminase having a cytidine deaminase function; the coding sequence of the TadA-CDa deaminase is shown in SEQ ID No.1.

The technical solution of the present invention is a fusion protein TadCBEa with cytosine base editing function, including TadA-CDa deaminase and Cas protein, with a structure of TadA-CDa deaminase-Cas protein or Cas protein-TadA-CDa deaminase; the TadA-CDa deaminase is TadA-8e with adenosine deaminase function modified to perform cytidine deaminase function.

Furthermore, the coding sequence of the TadA-CDa deaminase is shown in SEQ ID No.1.

Particularly, one or two UGIs are fused to either end of the fusion protein TadCBEa.

Specifically, the coding sequence of the fusion protein TadCBEa is shown in positions 2065 to 6810 of SEQ ID No.2.

The present invention also provides a cytosine base editing system derived from adenosine deaminase, comprising a fusion protein TadCBEa constructed by fusing TadA-CDa deaminase with Cas protein, wherein the structure of the fusion protein TadCBEa is TadA-CDa deaminase-Cas protein or Cas protein-TadA-CDa deaminase; the TadA-CDa deaminase is TadA-8e with adenosine deaminase activity modified to perform cytidine deaminase function.

Furthermore, the coding sequence of the TadA-CDa deaminase is shown in SEQ ID No.1.

Particularly, the structure of the TadCBEa is TadA-CDa-Cas-2×UGI.

Furthermore, the structure of the TadCBEa is promoter TadA-CDa-Cas-2×UGI-terminator.

Furthermore, NLS is provided between the promoter, TadA-CDa, Cas, 2×UGI and terminator.

Wherein, the Cas is Cas9, Cas12a or Cas12b.

Specifically, the Cas9 is nCas9.

Among them, the cytosine base editing system also includes a crRNA transcription expression unit.

Specifically, the structure of the cytosine base editing system is a fusion protein TadCBEa-crRNA transcription expression unit or a crRNA transcription expression unit-fusion protein TadCBEa.

Furthermore, the crRNA transcription expression unit structure is promoter-crRNA scaffold-terminator.

Particularly, the promoter is OsUbil, ZmUbil or P35S.

Wherein, the terminator is pinII, AtHSP, NOS or T35S.

Preferably, the nucleotide sequence of the fusion protein TadCBEa-crRNA transcription expression unit is shown in SEQ ID No.2.

The present invention also provides a vector, a cell or a host for expressing the TadA-CDa deaminase or the fusion protein TadCBEa.

Furthermore, the vector is a plant expression vector.

The present invention also provides a vector, a cell or a host containing the cytosine base editing system.

Furthermore, the vector is a plant expression vector.

Beneficial effects of the present invention: The present invention engineers the adenosine deaminase TadA-8e, and transforms TadA-8e, which has the function of adenosine deaminase, into TadA-CDa, which functions as a cytidine deaminase. The TadA-CDa deaminase is fused with the Cas protein to construct the cytosine base editor TadCBEa. Unlike the adenine base editor ABE8e fused with the adenosine deaminase TadA-8e, which performs the base editing function from A to G, TadCBEa can realize the base editing function from C to T. The active nucleotide of the deaminase is converted from adenosine A to cytidine C, but its editing window and editing specificity have not changed, that is, TadCBEa has the same editing window and high editing specificity as ABE8e. Compared with the most mainstream cytosine base editor A3A_Y130F-CBE, the editing efficiency of TadCBEa from C to T in rice cells is comparable to that of A3A_Y130F-CBE. At the same time, no off-target effects caused by TadCBEa were detected in the rice genome and transcriptome. It has higher editing specificity than A3A_Y130F-CBE and is a highly active and specific cytosine base editing tool. The protein molecular weight of TadA-CDa is 166 amino acids, which is smaller than the 199 amino acids of A3A_Y130F, which is conducive to the delivery of expression vectors to target species and the occurrence of subsequent editing events. TadCBEa can achieve effective base editing from C to T and almost no off-target editing can be detected at the whole genome DNA level and transcriptome RNA level. The TadCBEa provided by the present invention expands the base editing tool library of plants and has good application prospects in plant germplasm resource development and gene function research. The application of TadCBEa will provide more options for basic biological and medical research.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram of the vector of TadCBEa, with A3A_Y130F-CBE and ABE8e as control groups. A3A_Y130F-CBE: cytosine base editor containing cytidine deaminase APOBEC3A_Y130F; ABE8e: adenine base editor containing adenosine deaminase TadA-8e; TadCBEa: cytosine base editor containing TadA-CDa; A3A_Y130F-CBE, ABE8e and TadCBEa base editing vectors: two expression vectors were designed for 20 endogenous rice sites, each targeting 10 sgRNA sites; pZmUbi1: maize Ubiquitin 1 promoter; NLS: nuclear localization signal; nCas9: single-stranded inactivated Streptococcus pyogenes-derived Cas9 protein gene; UGI: uracil DNA glycosylase inhibitor; tHSP: Arabidopsis HSP terminator; pOsUbi1: rice Ubiquitin 1 promoter; tRNA: transfer RNA gene; sgRNA: single guide RNA; tpinⅡ: pinⅡ terminator.

Figure 2. Results of base editing activity detection of TadCBEa at 20 endogenous sites based on rice protoplast transient transformation system. A: C to T editing efficiency, A to G editing efficiency and Indel efficiency of different base editing systems at 20 target sites; WT: not transferred into expression vector; A3A_Y130F: transferred into A3A_Y130F-CBE expression vector; ABE8e: transferred into ABE8e expression vector; TadCBEa: transferred into TadCBEa expression vector. B: Summary of editing efficiency of 20 endogenous sites, C to T editing efficiency and Indel efficiency in rice cells treated with four different methods: WT, A3A_Y130F-CBE, ABE8e and TadCBEa.

Figure 3. Editing windows of base editing from C to T and from A to G in the sgRNA region in different base editing systems based on rice protoplast transformation. A3A_Y130F: Editing window in rice cells transiently transformed by rice protoplasts after being transferred into the A3A_Y130F-CBE expression vector; ABE8e: Editing window in rice cells transiently transformed by rice protoplasts after being transferred into the ABE8e expression vector; TadCBEa: Editing window in rice cells transiently transformed by rice protoplasts after being transferred into the TadCBEa expression vector.

Figure 4. Evaluation of editing efficiency of different base editing systems in stable transformed rice plants based on rice stable transformation and rice whole genome sequencing. A: Schematic diagram of the expression vector of TadCBEa, pZmUbi1: maize Ubiquitin 1 promoter; TadA-CDa: variant of TadA-8e adenosine deaminase; NLS: nuclear localization signal; nCas9: single-stranded inactivated Cas9 protein gene from Streptococcus pyogenes; UGI: uracil glycosylase inhibitor; tHSP: Arabidopsis HSP terminator; pOsUbi1: rice Ubiquitin 1 promoter; tRNA: transfer RNA gene; sgRNA: single guide RNA; tpinⅡ: pinⅡ terminator. B: Experimental flow chart of Sanger sequencing and whole genome sequencing of regenerated plants and analysis flow chart of sequencing data. CK: control group; Editor: base editor; Tissue (with Agro): transgenic rice plants without deaminase, Cas9 protein and sgRNA expression frame, as tissue culture control; A3A_Y130F-CBE: stably transformed rice plants transformed with A3A_Y130F-CBE base editing system; TadCBEa: stably transformed rice plants transformed with TadCBEa base editing system; Raw Reads: raw data of whole genome sequencing; BWA: a software name for analyzing sequencing results. C: C to T editing efficiency and homozygous C to T editing efficiency in Tissue, ABE8e and TadCBEa stably transformed rice plants. D: Sequence display of different target sites of TadCBEa stably transformed rice plants after C to T base editing.

Figure 5. Analysis of TadCBEa editing specificity based on rice stable transformation, whole genome sequencing, and transcriptome sequencing. A: Flowchart of rice stable transformation, sample and sequencing data analysis flow chart for whole genome sequencing and transcriptome sequencing. B: Based on whole genome sequencing data, the number of Indels in Tissue, A3A_Y130F, and TadCBEa stably transformed rice plants analyzed by comparison with the wild-type rice genome. C: The number of SNVs in Tissue, A3A_Y130F, and TadCBEa stably transformed rice plants. D: The number of different types of SNVs in Tissue, A3A_Y130F, and TadCBEa stably transformed rice plants. E: The number of C to T SNVs in Tissue, A3A_Y130F, and TadCBEa stably transformed rice plants. F: The proportion of different types of SNVs in Tissue, A3A_Y130F, and TadCBEa stably transformed rice plants to all SNVs. G: Editing motif preference of the TadCBEa base editing system in TadCBEa-stably transformed rice plants.

Figure 6. Detection of off-target effects dependent on sgRNA sequence in TadCBEa stably transformed rice plants based on rice stable transformation and rice whole genome sequencing. A: TadCBEa_OsALS: off-target analysis of TadCBE stably transformed rice plants at the target site OsALS; target gRNA: targeted gRNA sequence; allele1: allele 1; allele2: allele 2. B: TadCBEa_OsSPL14: off-target analysis of TadCBE stably transformed rice plants at the target site OsSPL14.

Figure 7. Analysis of TadCBEa editing specificity based on rice stable transformation and transcriptome sequencing. A: Number of SNVs at the RNA level in rice plants stably transformed with tissue, A3A_Y130F, and TadCBEa. B: Number of C to U SNVs at the RNA level in rice plants stably transformed with tissue, A3A_Y130F, and TadCBEa. C: Proportion of different types of SNVs at the RNA level in rice plants stably transformed with tissue, A3A_Y130F, and TadCBEa in total SNVs.

Detailed ways

In order to expand the CBE tool library in plants and discover CBEs with better editing efficiency and editing specificity, the present invention optimizes the rice codon preference of the evolved cytidine deaminase TadA-CDa, detects the base editing characteristics of TadCBEa in rice cells, and performs whole genome sequencing and transcriptome sequencing on the regenerated seedlings obtained by stable transformation of rice to evaluate the editing efficiency and editing specificity of TadCBEa in stable transformation regenerated plants. The application of TadCBEa will provide more options for basic biological and medical research.

As mentioned in the background introduction above, there are currently a variety of plant cytosine base editors available, including rAPOBEC1, PmCDA1, hAPOBEC3A, etc., which can achieve base editing from C to T at specific sites in the plant genome, but current cytosine base editors such as rAPOBEC1-CBE will produce off-target mutations at non-target sites (jin et al., Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. 2019, Science), which leads to genome safety issues. The adenine base editor currently used is mainly TadA-8e, which can achieve base editing from A to G at the target site and has high editing specificity.

Considering that both cytidine deaminase and adenosine deaminase perform the function of nucleoside deamination, is it possible to direct the evolution of adenosine deaminase with higher specificity into cytidine deaminase with higher editing specificity, so as to obtain a cytosine base editor with better editing performance. At present, studies have shown that through the directed evolution of adenosine deaminase TadA-8e, an adenosine deaminase variant TadA-CDa with good editing performance in human cells was obtained. The directed evolution product TadA-CDa has the characteristics of cytidine deaminase, so it can be classified as a type of cytidine deaminase. In order to expand the library of plant base editing tools and obtain cytosine base editors with higher editing activity and higher editing specificity, the cytidine deaminase TadA-CDa, which performs well in human cells, was fused with Cas9 protein to obtain TadA-derived Cytosine Base Editor a (TadCBEa). The editing activity and editing specificity of TadCBEa in rice cells and stable rice plants were then systematically evaluated in rice cells, providing a powerful tool with higher editing activity and higher editing specificity for plant base editing.

On the basis of the above, the inventors first optimized the codon preference of the coding sequence of cytidine deaminase TadA-CDa, taking into account the differences between species and the differences between plants and human cells in transcriptional translation expression systems, and obtained a TadA-CDa coding sequence (Seq ID No. 1) that is more suitable for plant species (especially Gramineae, such as rice, corn, sorghum, barley, wheat, sugarcane, etc.). Then, the coding sequence of TadA-CDa was combined with the Cas9 protein sequence, UGI sequence, and sgRNA expression frame into an expression vector, and the editing performance of TadCBEa was tested in rice protoplast cells and rice plants, respectively. Its editing efficiency is comparable to the best existing A3A_Y130F-CBE cytosine base editor, and its editing specificity is significantly better than A3A_Y130F-CBE.

The present invention will be further described below in conjunction with examples. The following examples are intended to illustrate the present invention rather than to further limit the present invention, and should not be used to limit the scope of protection of the present invention.

Example 1 Construction of TadCBEa cytosine base editing system vector

1. Optimization and synthesis of TadA-CDa coding sequence

Referring to the TadA-CDa coding sequence published by Neugebauer et al. in Nature Biotechnology in 2022 (Neugebauer et al., Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. 2022, Nature Biotechnology), we optimized it according to the codon preference of rice (Seq ID No. 1) to make it more suitable for expression in rice. The codon-optimized TadA-CDa coding sequence was commissioned to Qingke Company for DNA sequence synthesis.

Seq ID No.1TadA-CDa nucleotide sequence after optimization of rice codon preference;

TCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGGCACGGGATGAAGGCGCGGGGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTGGTCATGCAGAACTACAGACTCTTTGACGCCACCCTGTACGTGACATTC GAGCCTTGCGTGATGTGCGCCGGCGCCATGATCAACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACTCAAAAAGAGGCGCCGCAGGCTCCCTGATGAACGTGCTGAACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTGCCGCCCTGCTGTGCGATTTCTATCGGATGCCTAGACAGGTGTTCAACAGCCAGAAGAAGGCCCAGAGCTCCATCAAC.

2. Construction of TadA-CDa-Cas9-UGI expression cassette

pTSWA is the backbone vector of the TadA-Dual-Cas9-UGI expression cassette. Its function is to provide the overhang sequence that connects the TadA-Dual-Cas9-UGI expression cassette to the T-DNA backbone vector and the sgRNA expression cassette. The overhang sequence of the 5' end connected to the T-DNA backbone vector pTX1500 is GATC, and the overhang sequence of the 3' end connected to the sgRNA expression cassette is GGAC. Using pTSWA as the entry vector, the ZmUbi1 promoter, TadA-CDa coding sequence, Cas9 protein gene, UGI coding sequence and AtHsp terminator are cloned into pTSWA in sequence through Golden Gate mediated by BsaI enzyme and T4 DNA ligase to complete the assembly of the TadA-CDa-Cas9-UGI expression cassette. Golden Gate reaction system: T4 DNA ligase buffer, 2 μL; T4 DNA ligase, 1 μL; BsaI enzyme, 1 μL; pTSWA, 0.5 μL; ZmUbi1 promoter, 1 μL; TadA-CDa expression element, 3 μL; nCas9 expression element, 2.5 μL; UGI expression element, 2 μL; AtHsp terminator, 1 μL; ddH ₂ O, 6 μL. Golden Gate reaction program: (37°C, 5 min; 16°C, 10 min) × 15 cycles; 37°C, 10 min; 65°C, 10 min; 12°C, 10 min. After the ligation reaction is completed, take 10 μL of Golden Gate ligation product to transform Escherichia coli DH5α competent cells, spread on LB solid medium containing 50 mg/L Spec antibiotics, and culture at 37°C for 16 hours. Pick a monoclonal colony on the plate and dilute it in 50 μL sterile deionized water, take 5 μL of bacterial solution as a template, and use oCS433 (5'-GCTCACATGTTCTTTCCTGCG-3') and TX412 (5'-CCCTAAACCCTAAATGGATGTACTA-3') as primers for colony PCR. The length of the positive amplification product is 533 bp. The sequence of the vector was further verified by restriction digestion and sequencing, and the TadA-CDa-nCas9-UGI expression frame was constructed. As a control, the TadA-CDa coding sequence was replaced by the A3A_Y130F deaminase sequence and the TadA-8e deaminase sequence in A3A_Y130F-CBE and ABE8e, respectively. The subsequent vector construction steps are consistent with those of TadCBEa.

3. Construction of sgRNA expression cassette

In order to comprehensively detect the editing activity of TadCBEa base editor in rice, 20 targeting sgRNA sites were designed for rice endogenous genes. Every 10 sgRNAs formed a gRNA array. The site sequences are shown in Table 1, and the primers used to construct the sgRNA expression frame are shown in Table 2. The construction of the sgRNA expression frame is described below using the construction of gRNA array 01 as an example.

Table 1 Targeting sgRNA sequences and uses in sgRNA expression frames

Targeting sgRNA Targeted genes sgRNA sequence (5'-3') PAM Sequence use OsCGRS55-sgRNA01 OsCGRS55 CCACCCCTCCATCTCCTCCA CGG gRNA Array 01 OsAAT-sgRNA01 OsAAT CAAGGATCCCAGCCCCGTGA AGG gRNA Array 01 OsACC-sgRNA01 OsACC CATAGCACTCAATGCGGTCT GGG gRNA Array 01 OsACTG-sgRNA01 OsACTG ATCATCCGCCACGACGGCGG CGG gRNA Array 01 OsALS-sgRNA01 OsALS CGCGTCCATGGAGATCCACC AGG gRNA Array 01 OsCDC48-sgRNA01 OsCDC48 GACCAGCCAGCGTCTGGCGC CGG gRNA Array 01 OsD18-sgRNA01 OsD18 AGCAACCATTCATGGATGGA TGG gRNA Array 01 OsDEP1-sgRNA01 OsDEP1 AGACAAGCTTGGCCCTCTTT GGG gRNA Array 01 OsEV-sgRNA01 OsEV CACACACACACTAGTACCTC TGG gRNA Array 01 OsGRF1-sgRNA01 OsGRF1 GAACCGTTCAAGAAAGCCTG TGG gRNA Array 01 OsCGRS56-sgRNA01 OsCGRS56 CCCAACCCCCACTGACACCT AGG gRNA array 02 OsAAT-sgRNA02 OsAAT CTGCGCCCCGCGCACGTTAG CGG gRNA array 02 OsACC-sgRNA02 OsACC TCCACAGCTATCACACCCAC TGG gRNA array 02 OsALS-sgRNA02 OsALS TCTCCATGGACGCGCCGCCC GGG gRNA array 02 OsCDC48-sgRNA02 OsCDC48 TAGCACCCATGACAATGACA TGG gRNA array 02 OsDEP1-sgRNA02 OsDEP1 AGCACATGAGAGAACAATAT TGG gRNA array 02 OsGS3-sgRNA02 OsGS3 cTACGCGCATCTCTATATTT CGG gRNA array 02 OsLDMAR-sgRNA01 OsLDMAR GTGCATCCTGGGAAAGCTTG TGG gRNA array 02 OsPDS-sgRNA01 OsPDS GTTGGTCTTTGCTCCTGCAG AGG gRNA array 02 OsSPL14-sgRNA01 OsSPL14 TCTTCTGTCAACCCAGCCAT GGG gRNA array 02

Table 2 Primers used to construct sgRNA expression cassettes and their uses

pTX1290 sequentially contains the overhang sequence GGAC connected to the TadA-Dual-Cas9-UGI expression frame at the 5' end, the OsUbi1 promoter sequence, the tRNA sequence, the lacZα sequence, the sgRNA scaffold sequence, the tRNA sequence, the pinII terminator sequence, and the overhang sequence CCGG connected to the pMOD_C0000a vector at the 3' end. pMOD_C0000a acts as a linker between the sgRNA expression frame and the T-DNA backbone vector pTX1500. Its 5' end is the overhang sequence CCGG connected to the sgRNA expression frame, and its 3' end is the overhang sequence AGTG connected to the T-DNA backbone vector pTX1500. During the assembly of the gRNA array into the sgRNA expression frame, the PCR amplification product containing the sgRNA-sgRNA scaffold-tRNA sequence replaces the lacZα sequence therein through the Golden Gate ligation reaction, and multiple sgRNA-sgRNA scaffold-tRNA sequences are connected in series to form an sgRNA expression frame targeting multiple sgRNAs.

First, the sgRNA expression frame intermediate vector is constructed. Using pTX1290 as PCR template, primers FT188-F1 and FT188-R1 were used to amplify FT252-frag01, primers FT188-F2 and FT188-R2 were used to amplify FT252-frag02, primers FT188-F3 and FT188-R3 were used to amplify FT 252-frag03, and primers FT188-F4 and FT188-R4 were used to amplify FT252-frag04. The amplified products were purified by agarose gel electrophoresis and then recovered. The recovered amplified products and TA Blunt Vector were subjected to Golden Gate ligation reaction. The Golden Gate reaction system included: T4 DNA ligase buffer, 2 μL; T4 DNA ligase, 1 μL; BpiI enzyme, 1 μL; Bsp1407I enzyme, 1 μL; TA Blunt Vector, 1 μL; FT252-fra g, 1 μL each, a total of 4 μL; ddH ₂ O, 10 μL. Golden Gate reaction program: (37℃, 5min; 16℃, 10min) × 15 cycles; 37℃, 10min; 65℃, 10min; 12℃, 10min. After the ligation reaction was completed, 10μL of Golden Gate ligation product was transformed into E. coli DH5α competent cells, spread on LB solid medium containing 100mg/L Amp antibiotics, and cultured at 37℃ for 16 hours. Pick the monoclonal colony on the plate and dilute it to 50μL sterile deionized water, take 5μL of bacterial solution as a template, and use M13-F (5'-TGTAAAACGACGGCCAGT-3') and M13-R (5'-CAGGAAACAGCTATGAC-3') as primers for colony PCR. The length of the positive amplification product was 1515bp. The sequence of the vector was further verified by restriction digestion and sequencing, and the construction of the intermediate vector 01 of sgRNA array 01 was completed. Similarly, primer FT189 was used to construct intermediate vector 02, and primer FT190 was used to construct intermediate vector 03.

Then the sgRNA expression frame was constructed. Using pTX1290 as the entry vector, the intermediate vectors 01, 02 and 03 were connected in sequence through Golden Gate. Golden Gate reaction system: T4 DNA ligase buffer, 2μL; T4 DNA ligase, 1μL; BsaI enzyme, 1μL; pTX1290, 1μL; intermediate vector, 2μL each, 6μL in total; ddH ₂ O, 9μL. Golden Gate reaction program: (37℃, 5min; 16℃, 10min)×15 cycles; 37℃, 10min; 65℃, 10min; 12℃, 10min. After the ligation reaction is completed, take 10μL of Golden Gate ligation product to transform Escherichia coli DH5α competent cells, spread on LB solid medium containing 50mg/L Spec antibiotics, and culture at 37℃ for 16 hours. Pick the monoclonal colony on the plate and dilute it in 50 μL sterile deionized water, take 5 μL of bacterial solution as a template, and use TX421 (5'-TAATTTGATTGACTGATTTCTGCTGTA-3') and oCS1185 (5'-TGCATTACAGCTTACGAACC GAAC-3') as primers for colony PCR. The length of the positive amplification product is 2966 bp. The sequence of the vector was further verified by restriction digestion and sequencing, and the sgRNA expression frame of sgRNA array 01 was constructed. sgRNA array 01 is based on pTX1290 and 10 (sgRNA sequence-sgRNA scaffold sequence-tRNA sequence) are connected, including OsUbi1 promoter sequence, tRNA sequence, (sgRNA sequence-sgRNA scaffold sequence-tRNA sequence) × 10 and pinII terminator sequence.

Finally, the vector of sgRNA array 02 was constructed according to the construction method of sgRNA array 01.

4. Construction of expression vector for TadCBEa base editing system

The T-DNA backbone vector pTX1500 sequentially contains a poly-A termination signal, a hygromycin resistance gene, a 35S promoter, an overhang sequence GATC connected to the TadA-Dual-Cas9-UGI expression frame, a CcdB lethal gene, and an overhang sequence AGTG connected to pMOD_C0000a. Using pTX1500 as the entry vector, the TadA-CDa-Cas9-UGI expression cassette and the sgRNA expression cassette were cloned into the pTX1500 entry vector in sequence through the GoldenGate reaction mediated by AarI enzyme and T4 DNA ligase. The Golden Gate reaction system was as follows: T4 DNA ligase buffer, 2 μL; T4 DNA ligase, 1 μL; AarI enzyme, 0.5 μL; 50×Oligo, 0.4 μL; pTX1500, 0.5 μL; TadA-CDa-nCas9-UGI expression cassette, 1 μL; sgRNA expression cassette, 1 μL; pMOD_C0000a, 0.5 μL; ddH ₂ O, 13.1 μL. The Golden Gate reaction program was as follows: (37°C, 5 min; 16°C, 10 min) × 15 cycles; 37°C, 10 min; 65°C, 10 min; 12°C, 10 min. After the ligation reaction was completed, 10 μL of Golden Gate ligation product was transformed into E. coli DH5α competent cells, spread on LB solid medium containing 50 mg/L Kan antibiotics, and cultured at 37°C for 16 hours. Pick the monoclonal colony on the plate and dilute and mix it with 50 μL sterile deionized water. Take 5 μL of bacterial solution as a template, and use TX421 (5'-TAATTTGATTGACTGATTTCTGCTGTA-3') and ZY065-RB (5'-TTCTAATAAACGCTCTTTTCTCT-3') as primers for colony PCR. The length of the positive amplification product was 1549 bp. The constructed expression vector was verified by ScaI restriction enzyme digestion and Sanger sequencing to verify the accuracy of the vector sequence, and the TadCBEa expression vector was constructed (Figure 1 and Seq ID No. 2).

Seq ID No.2Nucleotide sequence of TadA-CDa-nCas9-2×UGI backbone vector. Among them, 1-1998 is the maize Ubi1 promoter; 2044-2064 is the NLS sequence; 2065-2562 is the TadA-CDa deaminase sequence; 2563-2654 is the 32AA linker sequence; 2665-2685 is the NLS sequence; 2710-6810 is the nCas9 coding sequence; 6811-6858 is the NLS sequence; 6895-7422 is the 2×UGI sequence; 7500-7749 is the Arabidopsis HSP terminator; 7776-9306 is the rice Ubi1 promoter; 9315-9391 is the Gly-tRNA sequence; 9399-9806 is the LacZα coding sequence; 9958-10053 is the sgRNA scaffold sequence; 10054-10130 is the Gly-tRNA sequence; 10141-10449 is the pinII terminator;

CTGCAGTGCAGCGTGACCCGGTCGTGCCCCTCTCTTGAGATAAGGTGAGCATTGCATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTGCAGTTTATCTATCTTTATACATATATTTAAACTTTACTCTACGAATAATATAATCTATAGTACTACAATAATATCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGACATGGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTAGTGTGCATGTGTTCTCCTTTTTTTTTGCAAATAGCTTCACCTATAT AATACTTCATCCATTTTATTAGTACATCCATTTAGGGTTTAGGGTTAATGGTTTTTATAGACTAATTTTTTTAGTACATCTATTTTATTCTATTTTAGCCTCTAAATTAAGAAAACTAAAACTCTATTTTAGTTTTTTTATTTAATAATTTAGATATAAAATAGAATAAAATAAAGTGACTAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTTGTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACACCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAG CGAAGCAGACGGCACGGCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTCGGCATCCAGAAATTGCGTGGCGGAGCGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAATAAATAGACACCCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACACACAACCAGAACTCCCCCAAATCCACCCGTCGGCACCTCCGC TTCAAGGTACGCCGCTCGTCCTCCCCCCCCCCCCTCTCTACCTTCTCAAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTTGTGTTAGATCCGTGCTACTAGCGTTCGTACACGGATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGGAATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTGTTTCGTTGCATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCA CTTGTTTGTCGGGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGTTCAAGATCGGAGTAGAATTAATTCTGTTTCAAACTACCTGGTGGATTTATTAATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGTTTTACTGATGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGGGCGGTCGTTCATTCGTTCAAGATCGGAGTAGAATACT GTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTTTTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATTAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATATACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCATACGCTATTTATTTGCTTGGTACTGTTTCTTT TGTCGATGCTCACCCTGTTGTTTGGTGTTACTTCTGCAGCCTGCAGGAATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGGCACGGGATGAAGGCGCGGGGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTGGTCATGCAGAACTACAGACT CTTTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCGGCGCCATGATCAACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACTCAAAAAGAGGCGCCGCAGGCTCCCTGATGAACGTGCTGAACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTGCCGCCCTGCTGTGCGATTTCTATCGGATGCCTAGACAGGTGTTCAACAGCCAGAAGAAGGCCCAGAGCTCCATCAACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCACAAG CGAGAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGTTCGATGGCTCCGAAGAAGAAGAGGAAGGTTGGCATCCACGGGGTGCCAGCTGCTGACAAGAAGTACTCGATCGGCCTCGCTATTGGGACTAACTCTGTTGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCAAAGAAGTTCAAGGTCCTGGGCAACACCGATCGGCATTCCATCAAGAAGAATCTCATTGGCGCTCTCCTGTTCGACAGCGGCGAGACGGCTGAGGCTACGCGGCTCAAGCGCACCGCCCGCAGGCGGTACACGCGCAGGAAGAATC GCATCTGCTACCTGCAGGAGATTTTCTCCAACGAGATGGCGAAGGTTGACGATTCTTTCTTCCACAGGCTGGAGGAGTCATTCCTCGTGGAGGAGGATAAGAAGCACGAGCGGCATCCAATCTTCGGCAACATTGTCGACGAGGTTGCCTACCACGAGAAGTACCCTACGATCTACCATCTGCGGAAGAAGCTCGTGGACTCCACAGATAAGGCGGACCTCCGCCTGATCTACCTCGCTCTGGCCCACATGATTAAGTTCAGGGGCCATTTCCTGATCGAGGGGGATCTCAACCCGGACAATAGCGATGTTGACAAGCTGTTCATCC AGCTCGTGCAGACGTACAACCAGCTCTTCGAGGAGAACCCCATTAATGCGTCAGGCGTCGACGCGAAGGCTATCCTGTCCGCTAGGCTCTCGAAGTCTCGGCGCCTCGAGAACCTGATCGCCCAGCTGCCGGGCGAGAAGAAGAACGGCCTGTTCGGGAATCTCATTGCGCTCAGCCTGGGGCTCACGCCCAACTTCAAGTCGAATTTCGATCTCGCTGAGGACGCCAAGCTGCAGCTCTCCAAGGACACATACGACGATGACCTGGATAACCTCCTGGCCCAGATCGGCGATCAGTACGCGGACCTGTTCCTCGCTGCCAAGAAT CTGTCGGACGCCATCCTCCTGTCTGATATTCTCAGGGTGAACACCGAGATTACGAAGGCTCCGCTCTCAGCCTCCATGATCAAGCGCTACGACGAGCACCATCAGGATCTGACCCTCCTGAAGGCGCTGGTCAGGCAGCAGCTCCCCGAGAAGTACAAGGAGATCTTCTTCGATCAGTCGAAGAACGGCTACGCTGGGTACATTGACGGCGGGGCCTCTCAGGAGGAGTTCTACAAGTTCATCAAGCCGATTCTGGAGAAGATGGACGGCACGGAGGAGCTGCTGGTGAAGCTCAATCGCGAGGACCTCCTGAGGAAGCAGCGGACA TTCGATAACGGCAGCATCCCACACCAGATTCATCTCGGGGAGCTGCACGCTATCCTGAGGAGGCAGGAGGACTTCTACCCTTTCCTCAAGGATAACCGCGAGAAGATCGAGAAGATTCTGACTTTCAGGATCCCGTACTACGTCGGCCCACTCGCTAGGGGCAACTCCCGCTTCGCTTGGATGACCCGCAAGTCAGAGGAGACGATCACGCCGTGGAACTTCGAGGAGGTGGTCGACAAGGGCGCTAGCGCTCAGTCGTTCATCGAGAGGATGACGAATTTCGACAAGAACCTGCCAAATGAGAAGGTGCTCCCTAAGCACTCGCT CCTGTACGAGTACTTCACAGTCTACAACGAGCTGACTAAGGTGAAGTATGTGACCGAGGGCATGAGGAAGCCGGCTTTCCTGTCTGGGGAGCAGAAGAAGGCCATCGTGGACCTCCTGTTCAAGACCAACCGGAAGGTCACGGTTAAGCAGCTCAAGGAGGACTACTTCAAGAAGATTGAGTGCTTCGATTCGGTCGAGATCTCTGGCGTTGAGGACCGCTTCAACGCCTCCCTGGGGACCTACCACGATCTCCTGAAGATCATTAAGGATAAGGACTTCCTGGACAACGAGGAGAATGAGGATATCCTCGAGGACATTGTGCTGAC ACTCACTCTGTTCGAGGACCGGGAGATGATCGAGGAGCGCCTGAAGACTTACGCCCATCTCTTCGATGACAAGGTCATGAAGCAGCTCAAGAGGAGGAGGTACACCGGCTGGGGGAGGCTGAGCAGGAAGCTCATCAACGGCATTCGGGACAAGCAGTCCGGGAAGACGATCCTCGACTTCCTGAAGAGCGATGGCTTCGCGAACCGCAATTTCATGCAGCTGATTCACGATGACAGCCTCACATTCAAGGAGGATATCCAGAAGGCTCAGGTGAGCGGCCAGGGGGACTCGCTGCACGAGCATATCGCGAACCTCGCTGGCTCGCC AGCTATCAAGAAGGGGATTCTGCAGACCGTGAAGGTTGTGGACGAGCTGGTGAAGGTCATGGGCAGGCACAAGCCTGAGAACATCGTCATTGAGATGGCCCGGGAGAATCAGACCACGCAGAAGGGCCAGAAGAACTCACGCGAGAGGATGAAGAGGATCGAGGAGGGCATTAAGGAGCTGGGGTCCCAGATCCTCAAGGAGCACCCGGTGGAGAACACGCAGCTGCAGAATGAGAAGCTCTACCTGTACTACCTCCAGAATGGCCGCGATATGTATGTGGACCAGGAGCTGGATATTAACAGGCTCAGCGATTACGACGTCGATCA TATCGTTCCACAGTCATTCCTGAAGGATGACTCCATTGACAACAAGGTCCTCACCAGGTCGGACAAGAACCGGGGCAAGTCTGATAATGTTCCTTCAGAGGAGGTCGTTAAGAAGATGAAGAACTACTGGCGCCAGCTCCTGAATGCCAAGCTGATCACGCAGCGGAAGTTCGATAACCTCACAAAGGCTGAGAGGGGCGGGCTCTCTGAGCTGGACAAGGCGGGCTTCATCAAGAGGCAGCTGGTCGAGACACGGCAGATCACTAAGCACGTTGCGCAGATTCTCGACTCACGGATGAACACTAAGTACGATGAGAATGACAAGC TGATCCGCGAGGTGAAGGTCATCACCCTGAAGTCAAAGCTCGTCTCCGACTTCAGGAAGGATTTCCAGTTCTACAAGGTTCGGGAGATCAACAATTACCACCATGCCCATGACGCGTACCTGAACGCGGTGGTCGGCACAGCTCTGATCAAGAAGTACCCAAAGCTCGAGAGCGAGTTCGTGTACGGGGACTACAAGGTTTACGATGTGAGGAAGATGATCGCCAAGTCGGAGCAGGAGATTGGCAAGGCTACCGCCAAGTACTTCTTCTACTCTAACATTATGAATTTCTTCAAGACAGAGATCACTCTGGCCAATGGCGAGATCC GGAAGCGCCCCCTCATCGAGACGAACGGCGAGACGGGGGAGATCGTGTGGGACAAGGGCAGGGATTTCGCGACCGTCAGGAAGGTTCTCTCCATGCCACAAGTGAATATCGTCAAGAAGACAGAGGTCCAGACTGGCGGGTTCTCTAAGGAGTCAATTCTGCCTAAGCGGAACAGCGACAAGCTCATCGCCCGCAAGAAGGACTGGGATCCGAAGAAGTACGGCGGGTTCGACAGCCCCACTGTGGCCTACTCGGTCCTGGTTGTGGCGAAGGTTGAGAAGGGCAAGTCCAAGAAGCTCAAGAGCGTGAAGGAGCTGCTGGGGATC ACGATTATGGAGCGCTCCAGCTTCGAGAAGAACCCGATCGATTTCCTGGAGGCGAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATTAAGCTCCCCAAGTACTCACTCTTCGAGCTGGAGAACGGCAGGAAGCGGATGCTGGCTTCCGCTGGCGAGCTGCAGAAGGGGAACGAGCTGGCTCTGCCGTCCAAGTATGTGAACTTCCTCTACCTGGCCTCCCACTACGAGAAGCTCAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTCGAGCAGCACAAGCATTACCTCGACGAGATCATTGAGCAGATTTCCGAG TTCTCCAAGCGCGTGATCCTGGCCGACGCGAATCTGGATAAGGTCCTCTCCGCGTACAACAAGCACCGCGACAAGCCAATCAGGGAGCAGGCTGAGAATATCATTCATCTCTTCACCCTGACGAACCTCGGCGCCCCTGCTGCTTTCAAGTACTTCGACACAACTATCGATCGCAAGAGGTACACAAGCACTAAGGAGGTCCTGGACGCGACCCTCATCCACCAGTCGATTACCGGCCTCTACGAGACGCGCATCGACCTGTCTCAGCTCGGGGGCGACAAGCGGCCAGCGGCGACGAAGAAGGCGGGGCAGGCGAAGAAGAAGAA GGCAGGATCTGGTGGCTCTGGCGGCTCCGGAGGAAGCACTAATCTCTCAGATATAATCGAGAAGGAAACTGGGAAACAGCTGGTTATACAAGAGAGCATTCTCATGCTTCCGGAGGAGGTGGAGGAGGTCATCGGCAACAAGCCTGAATCTGACATTCTTGTTCATACCGCGTACGACGAAAGCACGGATGAGAATGTCATGCTTTTGACTTCTGATGCACCTGAGTACAAGCCGTGGGCGTTGGTCATCCAGGACTCTAACGGTGAGAACAAAATCAAAATGCTATCCGGCGGGTCGGGGGGGTCAGGCGGTTCGACGAACCTGAG TGATATTATTGAGAAAGAGACTGGGAAGCAGTTGGTCATCCAAGAGAGCATCCTGATGCTCCCTGAAGAAGTGGAGGAGGTGATCGGCAACAAGCCCGAATCGGACATTCTAGTGCATACTGCTTATGACGAGTCTACCGACGAGAATGTGATGTTATTGACCTCCGATGCTCCAGAATACAAGCCATGGGCGTTAGTAATACAAGATTCAAACGGCGAGAATAAAATCAAGATGCTGTCGGGAGGTTCAAAGCGTACTGCTGATGGTTCAGAGTTTGAGCCCAAAAAGAAAAGAAAAGTGTGAGCTTCTCGAGTATATGAAGATG AAGATGAAATATTTGGTGTGTCAAATAAAAAGCTTGTGTGCTTAAGTTTGTGTTTTTTTCTTGGCTTGTTGTGTTATGAATTTGTGGCTTTTTCTAATATTAAATGAATGTAAGATCACATTATAATGAATAAACAAATGTTTCTATAATCCATTGTGAATGTTTTGTTGGATCTCTTCTGCAGCATATAACTACTGTATGTGCTATGGTATGGACTATGGAATATGATTAAAGATAAGCGCTGAGCTCGGACGGCGCCGGAGCACATCAGTCTCTGCACAAAGTGCATCCTGGGCTGCTTCAATTATAAAGCCCCATTCACCAC ATTTTGCTAGATAGTCGAAAAGCACCATCAATATTGAGCTTCAGGTATTTTTGGTTGTGTTGTGGTTGGATTGATTCTAATATATACCAAATCAATATAATTCACTACCAAAATATACCATAGCCATCACAACTTTATTAATTTTGGTAGCTTAAGATGGTATATATAATAACCAATTAACAACTGATTCTAATTTTACTACGGCCCAGTATCTACCAATACAAAACAACGAGTATGTTTTCTTCCGTCGTAATCGTACACAGTACAAAAAAACCTGGCCAGCCTTTCTTGGGCTGGGGCTCTCTTTCGAAAGGTCACAAAACGTAC ACGGCAGTAACGCCGCTTCGCTGCGTGTTAACGGCCACCAACCCCGCCGTGAGCAAACGGCATCAGCTTTCCACCTCCTCGATATCTCCGCGGCGCCGTCTGGACCCGCCCCCTTTCCGTTCCTTTCTTTCCTTCTCGCGTTTGCGTGGTGGGGACGGACTCCCCAAACCGCCTCTCCCTCTCTTTATTTGTCTATATTCTCACTGGGCCCCACCCACCGCACCCCTGGGCCCACTCACGAGTCCCCCCCTCCCCACCTATAAATACCCCACCCCCTCCTCGCCTCTTCCTCCATCAATCGAATCCCCAAAATCGCAGAGAAAAAAA AATCTCCCCTCGAAGCGAAGCGTCGAATCGCCTTCTCAAGGTATGCGATTTTCTGATCCTCTCCGTTCCTCGCGTTTGATTTGATTTCCCGGCCTGTTCGTGATTGTGAGATGTTGTTGTTAGTCTCCGTTTTGCGATCTGTGGTAGATTTGAACAGGGTTAGATGGGGTTCGCGTGGTATGCTGGATCTGTGATTATGAGCGATGCTGTTCGTGGTCCAAGTATTTATTGGTTCGGATCTAGAAGTAGAACTGTGCTAGGGTTGTGATTTGTTCCGATCTGTTCAATCAGTAGGATTTAGTCTCTGTTTTTCTCGTTGATCCAAG TAGCAGCTTCAGGTATATTTTGCTTAGGTTGTTTTTGATTCAGTCCCTCTAGTTGCATAGATTCTACTCTGTTCATGTTTAATCTAAGGGCTGCGTCTTGTTGATTAGTGATTACATAGCATAGCTTTCAGGATATTTTACTTGCTTATGCCTATCTTATCAACTGTTGCACCTGTAAATTCTAGCCTATGTTAATTAACCTGCCTTATGTGCTCTCGGGATAGTGCTAGTAGTTATTGAATCAGTTTGCCGATGGAATTCTAGTAGTTCATAGACCTGCAGATTATTTTTGTGAACTCGAGCACGGTGCGACTCTCTATTTTGTTA GGTCACTGTTGGTGTTGATAGGTACACTGATGTTATTGTGGTTTAGATCGTGTATCTAACATATTGGAATAATTTGATTGACTGATTTCTGCTGTACTTGCTTGGTATTGTTATAATTTCATGTTCATAGTTGCTGACCATGCTTCGGTAATTGTGTGTGCAGCCTGCAGGAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGGAGACCTTACAGTTGGACACAGGCCAACTTGTGAAGATTGCAAAACGTGGCGGCGTGACCGCAATGGAGGCAGTGCA TGCATCGCGCAATGCACTGACGGGTGCCCCCCTGGAGACGGGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAA TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCACCGGTGGTCTCAGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTTGCTGGAAACAGCAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGC TGGTGCAGGTACTCGAGCCTAGACTTGTCCATCTTCTGGATTGGCCAAGTTAATTAATGTATGAAATAAAAGGATGCACACATAGTGACATGCTAATCACTATAATGTGGGCATCAAAGTTGTGTGTTATGTGTAATTACTAATTATCTGAATAAGAGAAAGAGATCATCCATATTTCTTATCCTAAATGAATGTCACGTGTCTTTATAATTCTTTGATGAACCAGATGCATTTTATTAACCAATTCCATATACATATAAATATTAATCATATATAATTAATATCAATTGGGTTAGCAAAACAAATCTAGTCTAGGTGTGTTTTGC.

Example 2 TadCBEa activity detection based on protoplast transient expression

1. Preliminary detection of TadCBEa editing activity in rice cells

A3A_Y130F-CBE was used as a control for C to T editing efficiency, and ABE8e was used as a control for A to G editing efficiency. 20 rice endogenous sites were used to evaluate the base editing activity of TadCBEa. For each base editor, two multi-target T-DNA vectors were constructed, targeting 10 target sites in the rice genome (Table 1). Transient transformation of rice protoplasts was performed, and the editing performance of the base editing system was evaluated by next-generation sequencing (NGS) of PCR amplicons.

Through analysis of NGS data, it was found that TadCBEa was able to achieve efficient C to T base editing at all target sites in rice cells (Figure 2A). In a comprehensive analysis of all 20 targeted sites, the C to T base editing efficiency of TadCBEa was 4.5% to 55.4%, which was comparable to A3A_Y130F (9.0% to 53.0%) (Figure 2B). TadCBEa exhibited detectable A to G editing at some targeted sites (Figure 2A). In addition, the insertion and deletion (Indel) efficiencies of the TadCBEa base editor were very low and within the range of sequencing errors (Figure 2B). Taken together, these data indicate that TadCBEa has great prospects in achieving higher cytosine base editing efficiency and purity.

2. Base editing properties of TadCBEa in rice cells

Using NGS data, we further analyzed the editing features at the 20 targeted sites. Compared with the C to T editing window of A3A_Y130F spanning from position 3 to position 16, the C to T editing window of TadCBEa is narrower and concentrated from position 4 to position 8 of the protospacer, which is consistent with the A to G base editing window of ABE8e (Figure 3). The data show that the engineered TadCBEa, despite the change in base editing preference, still retains the base editing window of TadA-8e.

Example 3 Targeted modification of plant endogenous genes based on TadCBEa

1. Targeting multiple sites of TadCBEa for stable transformation of rice

The TadCBEa-gRNA array 02 vector (Figure 4A) was transferred into Agrobacterium tumefaciens EHA105 by freeze-thaw method, and the T-DNA backbone vector without deaminase and Cas protein and the A3A_Y130F-CBE-gRNA array 02 vector were transferred as tissue culture control and efficiency control respectively to perform experiments simultaneously and screen positive clones. The above vectors were transferred into rice callus by Agrobacterium-mediated genetic transformation method, and stably transformed transgenic plants were obtained in about 8 weeks (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. 1994, Plant Journal).

2. TadCBEa targeting multiple sites achieves stable editing in rice plants

First, the transgenic plants were subjected to Sanger sequencing to confirm that base editing events had indeed occurred at the target site, followed by whole genome sequencing (WGS) (Figure 4B). After aligning the WGS data with the reference rice genome, analysis of the editing efficiency in the plants showed that the C to T base editing efficiency of TadCBEa in stable plants was comparable to that of A3A_Y130F-CBE (Figure 4C), consistent with the data in protoplasts (Figure 2B). It is worth noting that TadCBEa found efficient biallelic C to T base editing at most target sites (Figure 4C). Further analysis of the target site sequences of the edited plants revealed that all base editing events in TadCBEa plants occurred within the editing window of TadCBEa (Figure 4D).

Example 4 Specificity analysis of plant endogenous gene base editing based on TadCBEa

1. Experimental design for TadCBEa editing specificity analysis

Whole genome sequencing and transcriptome sequencing were used to analyze the off-target effects in rice plants edited with TadCBEa multiple target sites. The off-target effects produced by gene editing tools are generally divided into off-target effects that are independent of sgRNA and off-target effects that are dependent on sgRNA. In the off-target analysis based on WGS and transcriptome sequencing, we used transgenic plants without T-DNA vectors for deaminase and Cas proteins as tissue culture controls, and used A3A_Y130F-CBE as a control for editing specificity (Figure 5A).

2. Analysis of off-target effects of TadCBEa independent of sgRNA based on whole genome sequencing

Analysis of WGS data revealed that TadCBEa produced similar numbers of insertions and deletions (Indels) (approximately 60) and single nucleotide variations (SNVs) (approximately 200) as tissue culture controls (Figures 5B and 5C). Further analysis of the six SNV types showed similar trends between TadCBEa base-edited plants and tissue culture control plants (Figure 5D). There was no significant increase in C to T SNVs in TadCBEa-edited plants compared with tissue culture controls, and the number of C to T SNVs in TadCBEa-edited plants was significantly reduced compared with A3A_Y130F-CBE, indicating that TadCBEa has better editing specificity (Figure 5E). Overall, TadCBEa and tissue culture controls shared similar SNV compositions (Figure 5F). Evaluation of the mutated cytosine-adjacent sequences did not reveal any sequence preference (Figure 5G), such as the known TA motif of TadA-8e. These analyses showed that TadCBEa had no significant genome-wide sgRNA-independent off-target effects in rice and that each mutation discovered by WGS was primarily due to somatic variation arising from tissue culture.

3. Analysis of off-target effects of TadCBEa dependent on sgRNA based on whole genome sequencing

Since base editing was performed simultaneously on 10 endogenous sites in the rice genome, we wanted to know whether any of the sgRNAs caused sgRNA-dependent off-target mutations. It is important to understand whether any of these SNVs are caused by off-target effects of the base editor. To this end, Cas-OFFinder (Bae et al., Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. 2014, Bioinformatics) was used to screen 20 potential off-target sites with ≤3 mismatched bases in the 10 target sites. Then, all C to T SNVs found in the edited plants were matched to these potential off-target sites. Analysis showed that 16 of the 20 potential off-target sites did not show detectable editing events in TadCBEa-edited plants (Table 3). Of the remaining 4 off-target sites, editing events at 3 off-target sites occurred at positions with one mismatched base with the target site (Figure 6 and Table 3). This suggests that the gRNA-dependent off-target mutations of these novel base editors are simply caused by the high sequence similarity between the target and off-target sites, which is also a feature of previous off-target effects analyzed by WGS based on Cas9 in plants.

Table 3 Potential off-target sites dependent on sgRNA and the proportion of plants with off-target

4. Analysis of off-target effects of TadCBEa at the RNA level based on transcriptome sequencing

Since TadA variants may cause transcriptome-wide off-target mutations in plants when overexpressed (Li et al., A large-scale genome and transcriptome sequencing analysis reveals the mutationlandscapes induced by high-activity adenine base editors in plants. 2022, Genome Biology), we performed an off-target effect analysis of TadCBEa at the transcriptome level (Figure 5A). Compared with tissue culture controls, there were no significant changes in the total number of SNVs and the number of C to U SNVs in TadCBEa-edited plants (Figures 7A and 7B), and the proportions of different types of SNVs remained unchanged in TadCBEa-edited plants (Figure 7C). This indicates that the base editor did not introduce additional C to U mutations in the rice transcriptome.

Overall, TadCBEa did not cause significant off-target effects at both the DNA and RNA levels, indicating that TadCBEa is a highly specific genomic base editing tool in plants.

Based on the results of the above embodiments, it can be seen that:

TadCBEa is an efficient cytosine base editor in rice cells with high editing purity while maintaining the relatively narrow editing window of ABE8e. TadCBEa can edit multiple target genes simultaneously in transgenic rice plants. In addition, no off-target effects of TadCBEa were detected by whole genome and transcriptome sequencing of transgenic plants, indicating its high editing specificity. In summary, this study reports a TadCBEa cytosine base editor derived from TadA-8e, which is a cytosine base editor with high editing activity, purity and specificity, and is a backup supplement in the plant gene editing toolbox.

Claims (12)

1. A TadA-CDa deaminase, characterized in that it has a deaminase function; the coding sequence of the TadA-CDa deaminase is shown in SEQ ID No.1. 2. A fusion protein TadCBEa with cytosine base editing function, characterized in that it includes TadA-CDa deaminase and Cas protein, and the structure is TadA-CDa deaminase-Cas protein or Cas protein-TadA-CDa deaminase; the coding sequence of the TadA-CDa deaminase is shown in SEQ ID No.1. 3. The fusion protein TadCBEa according to claim 2, characterized in that: one or two UGIs are also fused to either end of the fusion protein TadCBEa. 4. The fusion protein TadCBEa according to claim 2, characterized in that the coding sequence of the fusion protein TadCBEa is shown in positions 2065 to 6810 of SEQ ID No.2. 5. A cytosine base editing system derived from adenosine deaminase, characterized in that it comprises a fusion protein TadCBEa constructed by fusion of TadA-CDa deaminase and Cas protein, and the structure of the fusion protein TadCBEa is TadA-CDa deaminase-Cas protein or Cas protein-TadA-CDa deaminase; the coding sequence of the TadA-CDa deaminase is shown in SEQ ID No.1. 6. The cytosine base editing system according to claim 5, characterized in that the structure of the TadCBEa is TadA-CDa-Cas-2×UGI. 7. The cytosine base editing system according to claim 6, characterized in that it has one or more of the following features: a. The structure of TadCBEa is promoter-TadA-CDa-Cas-2×UGI-terminator; b. The Cas is Cas9, Cas12a or Cas12b; c. The cytosine base editing system also includes a crRNA transcription expression unit, and the structure of the cytosine base editing system is a fusion protein TadCBEa-crRNA transcription expression unit or a crRNA transcription expression unit-fusion protein TadCBEa. 8. The cytosine base editing system according to claim 7, characterized in that it has one or more of the following features: a. NLS is also provided between the promoter, TadA-CDa, Cas, 2×UGI and terminator; b. The Cas9 is nCas9; c. The crRNA transcription expression unit structure is promoter-crRNA scaffold-terminator. 9. The cytosine base editing system according to claim 7 or 8, characterized in that: the promoter is OsUbi1, ZmUbi1 or P35S; the terminator is pinII, AtHSP, NOS or T35S. 10. The cytosine base editing system according to claim 9, characterized in that the nucleotide sequence of the fusion protein TadCBEa-crRNA transcription expression unit is as shown in SEQ ID No.2. 11. A vector, cell or host expressing the TadA-CDa deaminase according to claim 1, the fusion protein TadCBEa according to any one of claims 2 to 4, or containing the cytosine base editing system according to any one of claims 5 to 10. 12. The vector, cell or host according to claim 11, characterized in that the vector is a plant expression vector.