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CN-121992018-A - Rice Sdd7-CGBE base editor and application thereof

CN121992018ACN 121992018 ACN121992018 ACN 121992018ACN-121992018-A

Abstract

The invention relates to the technical field of agriculture, in particular to a rice Sdd7-CGBE base editor and application thereof. The gene sequence of the rice Sdd7-CGBE base editor at least comprises (1) a nucleotide sequence shown as SEQ ID NO.1, or (2) a nucleotide sequence capable of carrying out rice genome cutting by replacing one or more nucleotide sequences in the nucleotide sequence shown as SEQ ID NO.1, or (3) a nucleotide sequence capable of carrying out rice genome cutting by adding one or more nucleotide sequences in the nucleotide sequence shown as SEQ ID NO.1, or (4) a nucleotide sequence capable of carrying out rice genome cutting by deleting one or more nucleotide sequences in the nucleotide sequence shown as SEQ ID NO. 1. The editor targets specific sites under nSpCas guidance through the efficient deamination activity of Sdd7 and uracil excision function of coUNG, and can reduce Indels and bystander effects while inducing C-G transversion.

Inventors

  • XU RONGFANG
  • LI JUAN
  • GU DONGFANG
  • QIN RUIYING

Assignees

  • 安徽省农业科学院水稻研究所

Dates

Publication Date
20260508
Application Date
20260204

Claims (10)

  1. 1. A rice Sdd7-CGBE base editor, wherein the gene sequence of the base editor comprises at least: (1) The nucleotide sequence shown as SEQ ID NO.1, or, (2) A nucleotide sequence capable of performing rice genome cleavage by substituting one or more nucleotide sequences in the nucleotide sequence shown in SEQ ID NO.1, or, (3) One or more nucleotide sequences capable of performing genome cleavage of rice are added to the nucleotide sequence shown in SEQ ID NO.1, or, (4) One or more nucleotide sequences are deleted in the nucleotide sequence shown in SEQ ID NO.1 and a nucleotide sequence capable of performing genome cleavage in rice.
  2. 2. The rice Sdd7-CGBE base editor of claim 1, wherein the gene sequence of the rice Sdd7-CGBE base editor is the nucleotide sequence shown in SEQ ID No. 1.
  3. 3. The rice Sdd7-CGBE base editor of claim 1, wherein the rice Sdd7-CGBE base editor comprises cytosine deaminase Sdd7, an inactivated nSpCas protein, and a codon-optimized uracil DNA glycosylase coUNG; the cytosine deaminase Sdd7 is positioned at the 5' end of the live nSpCas protein, and the cytosine deaminase Sdd7 and the live nSpCas protein are connected in series by a linker of 32 aa; The codon optimized uracil DNA glycosylase coUNG is positioned at the 3' end of the live nSpCas protein, and the two are connected in series by a linker of 32 aa.
  4. 4. An expression cassette comprising the rice Sdd7-CGBE base editor of any one of claims 1-3.
  5. 5. An expression vector comprising the rice Sdd7-CGBE base editor of any one of claims 1-3 or the expression cassette of claim 4.
  6. 6. The expression vector of claim 5, further comprising an sgRNA sequence and expression cassette that is adapted to the rice Sdd7-CGBE base editor.
  7. 7. The expression vector of claim 6, wherein the nucleotide sequence of the sgRNA sequence is set forth in SEQ ID No. 4.
  8. 8. Use of a rice Sdd7-CGBE base editor according to any one of claims 1-3, an expression cassette according to claim 4 or an expression vector according to any one of claims 5-7 to effect a rice base C-G inversion.
  9. 9. A method of constructing a fusion editing tool of a novel base editor Sdd7-CGBE system, comprising the steps of: The sgRNA expression cassette, cas9 protein, cytosine deaminase Sdd7 gene and uracil DNA glycosylase coUNG are introduced into a target receptor, and the sgRNA expression cassette, cas9 protein, cytosine deaminase Sdd7, uracil DNA glycosylase coUNG are expressed in the target receptor, thereby performing C-G base editing on the target gene in the genome of the receptor.
  10. 10. The method of constructing a rice base editing tool Sdd7-CGBE according to claim 7, wherein the cytosine dehydrogenase Sdd7 gene, cas9 protein, uracil DNA glycosylase coUNG uses the same constitutive promoter Ubiqutin and the same 35S terminator; the sgRNA expression frame comprises a rice OsU promoter, a spectinomycin resistance gene SpR, an artificially synthesized sgRNA skeleton sequence and a Poly-T terminator.

Description

Rice Sdd7-CGBE base editor and application thereof Technical Field The invention relates to the technical field of agriculture, in particular to a rice Sdd7-CGBE base editor and application thereof. Background Base editing is a technique that enables targeted single nucleotide mutation without breaking the DNA duplex. Currently the dominant tools include Cytosine Base Editors (CBEs), adenine Base Editors (ABEs) and guanine base editors (CGBEs). CBEs and ABEs mediate mainly transition mutations (e.g., c→t or a→g), whereas base transversions between pyrimidine and purine (e.g., c→g or a→t) cannot be directly achieved. It was found that after removal of Uracil Glycosylase Inhibitor (UGI) in CBE, partial editing sites were subjected to base transversion phenomena such as C.fwdarw.A and C.fwdarw.G. Based on this mechanism, several research teams in 2020 have engineered CBE's, respectively, to successfully develop a base editor GBE/CGBE that can achieve the C.fwdarw.G transversion in mammalian cells. CGBE is generally composed of three parts, nCas (D10A), cytidine deaminase and uracil-N-glycosylase (UNG), further expanding the range of application of single base editing tools. However, traditional CGBEs relies on natural cytosine deaminase (e.g., AID/apodec family) which is prone to activate the base excision repair pathway, leading to high frequency index and off-target effects. The specific mechanism is that UDG cuts uracil (U) to form abasic site (AP site), a single-chain gap is formed under the action of AP lyase or spontaneous cleavage, if the single-chain gap is coincident with a gap nCas generated by a non-editing chain, DNA double-chain cleavage is initiated, and indexes are generated through NHEJ repair. In addition, CBEs also present a significant DNA/RNA random off-target effect, with potential safety hazards. In the plant field, expanding the base substitution type has important significance for germplasm resource creation. Early-stage system based on first generation CGBE realizes C-G transversion in rice, tomato, aspen and other species, but has the problems of low efficiency, more byproducts and the like, and limits the wide application of the system. Disclosure of Invention In order to solve the technical problems, the invention provides a rice Sdd7-CGBE base editor and application thereof. The Sdd7-CGBE base editor is formed by introducing an sgRNA expression cassette, a Cas9 protein, a cytosine deaminase Sdd7 gene and uracil DNA glycosylase coUNG into a target receptor. The editor targets specific sites under nSpCas guidance through the efficient deamination activity of Sdd7 and uracil excision function of coUNG, and can reduce index and bystander effects while inducing C-G transversion. Therefore, the invention provides the following technical proposal, In a first aspect, the invention provides in an alternative embodiment a rice Sdd7-CGBE base editor, the gene sequence of which comprises at least: (1) The nucleotide sequence shown as SEQ ID NO.1, or, (2) A nucleotide sequence capable of performing rice genome cleavage by substituting one or more nucleotide sequences in the nucleotide sequence shown in SEQ ID NO.1, or, (3) One or more nucleotide sequences capable of performing genome cleavage of rice are added to the nucleotide sequence shown in SEQ ID NO.1, or, (4) One or more nucleotide sequences are deleted in the nucleotide sequence shown in SEQ ID NO.1 and a nucleotide sequence capable of performing genome cleavage in rice. Preferably, the gene sequence of the rice Sdd7-CGBE base editor is a nucleotide sequence shown as SEQ ID NO. 1. Preferably, the rice Sdd7-CGBE base editor comprises cytosine deaminase Sdd7, inactivated nSpCas protein and codon-optimized uracil DNA glycosylase coUNG; the cytosine deaminase Sdd7 is positioned at the 5' end of the live nSpCas protein, and the cytosine deaminase Sdd7 and the live nSpCas protein are connected in series by a linker of 32 aa; The codon optimized uracil DNA glycosylase coUNG is positioned at the 3' end of the live nSpCas protein, and the two are connected in series by a linker of 32 aa. In a second aspect, the invention provides in an alternative embodiment an expression cassette comprising a rice Sdd7-CGBE base editor as described above. In a third aspect, the invention provides in an alternative embodiment an expression vector comprising a rice Sdd7-CGBE base editor as described above or an expression cassette as described above. Preferably, the rice plant also comprises an sgRNA sequence and an expression frame which are matched with the rice Sdd7-CGBE base editor. Preferably, the nucleotide sequence of the sgRNA sequence is shown as SEQ ID NO. 4. In a fourth aspect, the invention provides in an alternative embodiment the use of a rice Sdd7-CGBE base editor as described above, an expression cassette as described above or an expression vector as described above for effecting a rice base C-G inversion. In a fifth aspect, the present invention