Cytosine deaminase SvSddA, base editor containing cytosine deaminase SvSddA and application

Abstract

The invention provides cytosine deaminase SvSddA, a base editor containing the cytosine deaminase SvSddA and application of the cytosine deaminase SvSddA, the amino acid sequence of the cytosine deaminase SvSddA is shown as SEQ ID NO.1, SddA which is not reported yet is found near a branch of high-activity protein, the cytosine deaminase SvSddA serves as candidate protein to be further subjected to structure comparison, and the cytosine deaminase SddA from Saccharothrix violaaceirubra is selected as one of final candidate cytosine deaminase for activity evaluation on the basis of a structural comparison result, the editing efficiency of SvSddA at four nuclear target sites in HEK293T cells is detected, and efficient C-T editing efficiency is displayed at the position of a target, so that CBE containing SvSddA has the advantages of high activity, small size and the like, and the CBE can be used for preparing the CBE. Compared with other existing CBE, the method has obvious advantages.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing, and specifically relates to cytosine deaminase SvSddA, a base editor containing the same, and its application, which can effectively realize the conversion of C to T in eukaryotic cell genes. Background Art

[0002] The CRISPR system can perform precise editing in genomic DNA, but it relies on the homologous recombination (HDR) repair pathway in cells and has extremely low efficiency.

[0003] Base editing technology developed based on the CRISPR / Cas9 system fuses nucleases that have lost their cleavage activity with different base deaminases to construct two base editing systems (BE): cytosine base editor (CBE) and adenine base editor (ABE). These two types of editors can respectively complete C>T (G>A) or A>G (T>C) substitutions at the target gene site without generating double-strand breaks in the DNA, ultimately achieving precise base editing.

[0004] The current CBE can convert C·G base pairs to T·A and consists of a cytidine deaminase fused to Cas9 and a uracil glycosylase inhibitor (UGI) domain. Summary of the Invention

[0005] The present invention aims to provide a novel cytosine deaminase for optimizing CBE. The novel cytosine deaminase is a single-stranded DNA deaminase (SddA) derived from Saccharothrix violaceirubra, named SvSddA. It was discovered based on bioinformatics analysis of existing single-base DNA deaminase (SddA) data. The amino acid sequence of SvSddA is shown in the attached table.

[0006] Another object of the present invention is to provide a polynucleotide sequence for expressing the above-mentioned SvSddA. The specific nucleotide sequence is shown in SEQ ID NO.2.

[0007] Another object of the present invention is to provide a fusion protein, the amino acid sequence of which is shown in SEQ ID NO.3, the fusion protein comprising a Cas enzyme domain, a cytosine deaminase domain and a UGI domain; the cytosine deaminase domain is SvSddA.

[0008] Another object of the present invention is to provide a polynucleotide sequence for expressing the above-mentioned fusion protein, the specific nucleotide sequence of which is shown in SEQ ID NO.4.

[0009] Another object of the present invention is to provide a recombinant expression vector comprising the polynucleotide sequence of the above fusion protein.

[0010] Another object of the present invention is to provide a genetically engineered host cell comprising the above-mentioned recombinant expression vector or a polynucleotide sequence of the fusion protein integrated into the genome, as well as sgRNA and AAV.

[0011] Another object of the present invention is to provide a cytosine single-base editor, which is obtained by integrating a polynucleotide sequence encoding the above-mentioned fusion protein into an expression vector.

[0012] Another object of the present invention is to provide the use of the above-mentioned cytosine base editor in the preparation of a reagent for mediating gene editing, which is used for performing gene editing, reducing off-target effects, improving targeted editing efficiency, or improving the fidelity of targeted editing.

[0013] Another object of the present invention is to provide a gene editing method, comprising using the cytosine base editor to mediate gene editing, injecting the polynucleic acid sequence encoding the cytosine base editor and the sgRNA into a receptor to perform gene editing, wherein the receptor includes: somatic cells or germ cells, and the germ cells include embryonic cells or fertilized eggs.

[0014] Another object of the present invention is a reagent or kit for gene editing, which includes the above-mentioned SvSddA or the above-mentioned cytosine base editor.

[0015] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein.

[0016] This study examined all reported DddA and SddA data, artificially categorized the highly active proteins within the dataset, and performed multiple sequence alignment to generate a seed dataset for HMMER search. Protein sequence results retrieved from the generated database were then streamlined and then subjected to multiple sequence alignment with the processed protein set to construct a phylogenetic tree. This led to the discovery of an unreported SddA near the branch of highly active proteins. This candidate protein was then further subjected to structural alignment. Based on the structural alignment results, SddA from Saccharothrix violaceirubra was selected as one of the final candidate cytosine deaminases for activity evaluation.

[0017] Specifically, the present invention detected the editing efficiency of SvSddA at four nuclear target sites in HEK293T cells, and showed efficient C to T editing efficiency at the target position.

[0018] From the above, it can be seen that the CBE containing SvSddA of the present invention has many advantages such as high activity and small size, and has obvious advantages over other existing CBEs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the predicted structure of cytosine deaminase SvSddA of the present invention; Figure 2 This is the sequencing peak diagram of the sgRNA-1 C to T mutation efficiency. The PAM sequence recognized by sgRNA-1 is GGG. The C to T mutation site is indicated by the black arrow. The mutation efficiency evaluated at the C4 to T site is 31%, and the mutation efficiency evaluated at the C6 to T site is 21%.

[0020] Figure 3 This is the sequencing peak diagram of the sgRNA-2 C to T mutation efficiency. The PAM sequence recognized by sgRNA-2 is GGG. The C to T mutation site is pointed by the black arrow. The mutation efficiency evaluated by the C5 to T site is 18%.

[0021] Figure 4 This is the sequencing peak diagram of the sgRNA-3C to T mutation efficiency. The PAM sequence recognized by sgRNA-3 is TGG. The C-to-T mutation site is indicated by the black arrow. The mutation efficiency evaluated by the C6 to T site is 12%.

[0022] Figure 5 This is the sequencing peak diagram of the sgRNA-4 C to T mutation efficiency. The PAM sequence recognized by sgRNA-4 is TGG. The C to T mutation site is indicated by the black arrow. The mutation efficiency of the C3 to T site is evaluated as 7%, and the mutation efficiency of the C6 to T site is evaluated as 7%. DETAILED DESCRIPTION

[0023] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0024] Example 1: Constructing SvCBE.

[0025] The amino acid sequence of SvSddA was confirmed by amino acid sequence alignment as shown in the attached table, and the predicted structure was as follows Figure 1 SvSddA was synthesized and cloned into the AncBE4max vector from which APOBEC was removed to obtain SvCBE.

[0026] Example 2: Construction of sgRNA vector.

[0027] According to the human gene sequence of the NCBI gene library (version number: GRCh38), the region targeted by the sgRNA for gene editing was selected according to the PAM sequence (NGG), and four sgRNA sequences targeting human genes were designed and synthesized, as follows: sgRNA1-F sequence: 5-ccggGAACACAAAGCATAGACTGC-3 (SEQ ID NO. 5); sgRNA1-R sequence: 5-aaacGCAGTCTATGCTTTGTGTTC-3 (SEQ ID NO. 6); sgRNA2-F sequence: 5-ccggGAGTCCGAGCAGAAGAAGAA-3 (SEQ ID NO.7); sgRNA2-R sequence: 5-aaacTTCTTCTTCTGCTCGGACTC-3 (SEQ ID NO. 8); sgRNA3-F sequence: 5-ccggGGAATCCCTTCTTGCAGCACC-3 (SEQ ID NO.9); sgRNA3-R sequence: 5-aaacGGTGCTGCAGAAGGGATTCC-3 (SEQ ID NO. 10); sgRNA4-F sequence: 5-ccggGGCCACACTAGCGTTGCTGC-3 (SEQ ID NO. 11); sgRNA4-R sequence: 5-aaacGCAGCAACGCTAGTGTGGCC-3 (SEQ ID NO. 12).

[0028] The four pairs of single-stranded sgRNA DNA sequences were annealed to form four oligonucleotide chains targeting different human gene sites. The four sgRNAs were then ligated into the linearized 74707 vector after enzyme digestion to obtain four sgRNA expression vectors targeting human nuclear genes.

[0029] Example 3: CBE transfected 293T cells.

[0030] After sequencing and verifying the constructed sgRNA expression vector, the target plasmid was extracted and purified by ethanol sodium acetate precipitation. SvCBE and each sgRNA expression vector were introduced into 293T cells by lipofectamine transfection. The culture medium was replaced after 12 hours of culture. After 72 hours of transfection, the genome of each group of cells was extracted, and PCR reaction was performed with specific primers. The obtained PCR product was sequenced by agarose gel electrophoresis. The editing of specific gene sites in 293T cells was evaluated by sequencing peak analysis. The specific editing efficiency peak is shown in the figure below. Figures 2 to 5 .like Figure 2 As shown, the PAM sequence recognized by sgRNA-1 is GGG, the C-to-T mutation site is indicated by the black arrow, the mutation efficiency evaluated at the C4 to T site is 31%, and the mutation efficiency evaluated at the C6 to T site is 21%. Figure 3 As shown, the PAM sequence recognized by sgRNA-2 is GGG, the C to T mutation site is pointed by the black arrow, and the mutation efficiency evaluated by the C5 to T site is 18%. Figure 4 As shown, the PAM sequence recognized by sgRNA-3 is TGG, the C to T mutation site is pointed by the black arrow, and the mutation efficiency of the C6 to T site is 12%. Figure 5 As shown, the PAM sequence recognized by sgRNA-4 is TGG, the C-to-T mutation site is indicated by the black arrow, the mutation efficiency evaluated by the C3to T site is 7%, and the mutation efficiency evaluated by the C6to T site is 7%.

[0031] Sequencing peak analysis showed that SvSddA has the ability to deaminase cytosine in 293T cells, and the CBE composed of its combination can cause C to T conversion in the targeted DNA sequence under the guidance of sgRNA.

[0032] The targeted editing efficiency of the cytosine base editor of the present invention is significantly improved.

[0033] In the present invention, the vectors of the present invention may contain a primer sequence, for example, a CAG promoter. Furthermore, any mammalian-expressing promoter, such as the EF1α promoter, which is generally considered equivalent to the CAG promoter, may also be used. Furthermore, mammalian tissue-specific promoters, such as the ICAM2 promoter, may also be used. The CAG promoter, as one type of gene expression promoter, is used to express foreign genes. A "promoter" generally serves as the origin of transcription and is located at the beginning of the DNA base sequence carrying the genetic information of the gene to be expressed, within a few hundred bases of the transcription origin. In eukaryotes, proteins known as transcriptional regulatory factors bind to the promoter region, thereby participating in the binding of RNA polymerase.

[0034] In the present invention, "sgRNA" refers to small guide RNA (sgRNA).

[0035] In the present invention, the cytosine base editor is also connected to the nuclear localization sequence; preferably, the N-terminus and / or the C-terminus are connected to the nuclear localization sequence. A linker sequence, such as a tag sequence, is also included between the cytosine base editor and the nuclear localization sequence. The cytosine deaminase and the nuclear localization sequence are also connected by a linker sequence, and the linker sequence can be any linker sequence that does not affect the functions of the two, for example, it can be a tag sequence or some flexible linker sequences known in the art. Suitable tags can be used in the present invention. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE or Ty1.

[0036] The cytosine deaminase of the present invention can be a recombinant protein, a natural protein, or a synthetic protein, preferably a recombinant protein. The protein of the present invention can be a naturally purified product, a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells).

[0037] The present invention may also include fragments, derivatives and analogs of the SvSddA described above. As used herein, the terms "fragment," "derivative," and "analog" refer to proteins that substantially retain the same biological function or activity as the SvSddA of the present invention. The protein fragments, derivatives, or analogs of the present invention may be proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or proteins having a substitution group in one or more amino acid residues, or proteins formed by the fusion of an additional amino acid sequence to this protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the protein, or a fusion protein). According to the definitions herein, these fragments, derivatives, and analogs fall within the scope known to those skilled in the art.

[0038] In the present invention, SvSddA may also include (but is not limited to): deletions, insertions, and / or substitutions of several amino acids (usually 1-20, more preferably 1-10, and even more preferably 1-8, 1-5, 1-3, or 1-2), as well as additions or deletions of one or several amino acids (usually within 20, preferably within 10, and more preferably within 5) at the C-terminus and / or N-terminus. For example, in the art, substitutions with amino acids having similar or similar properties generally do not alter the function of the protein. For another example, the addition of one or several amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of modified enzymes.

[0039] In the present invention, SvSddA also includes (but is not limited to) derivative proteins that retain their protein activity and have a sequence identity of more than 80%, preferably more than 85%, more preferably more than 90%, and further more preferably more than 95%, such as more than 98% or more than 99% with the amino acid sequence of SvSddA.

[0040] The present invention also provides a polynucleotide sequence encoding the SvSddA of the present invention or its conservative variant protein.

[0041] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0042] The polynucleotide encoding the mature SvSddA protein includes: a coding sequence encoding only the mature protein; a coding sequence for the mature protein and various additional coding sequences; a coding sequence for the mature protein (and optional additional coding sequences) and non-coding sequences. A "polynucleotide encoding a protein" may include a polynucleotide encoding the protein or a polynucleotide further including additional coding and / or non-coding sequences.

[0043] The full-length SvSddA nucleotide sequence of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the relevant nucleotide sequence disclosed in the present invention, particularly the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequence. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified in each amplification are spliced ​​together in the correct order. Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is generally done by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0044] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained. At present, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. The present invention also relates to vectors comprising the polynucleotides of the present invention, as well as host cells genetically engineered using the vectors of the present invention or modified enzyme coding sequences, and methods for producing the proteins of the present invention by recombinant technology.

[0045] Recombinant SvSddA can be expressed or produced using the polynucleotide sequences of the present invention through conventional recombinant DNA techniques. Generally, the following steps are involved: (1) transforming or transducing a suitable host cell with the SvSddA polynucleotide of the present invention, or a recombinant expression vector containing the polynucleotide; (2) culturing the host cell in a suitable culture medium; and (3) isolating and purifying the protein from the culture medium or cells.

[0046] The present invention also provides a cytosine base editor comprising the modified enzyme or its polynucleotide sequence. The other components of the cytosine base editor are known to those skilled in the art.

[0047] In the present invention, the SvSddA polynucleotide sequence or the cytosine base editor polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, as long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0048] Methods well known to those skilled in the art can be used to construct an expression vector containing the SvSddA polynucleotide sequence or the cytosine base editor polynucleotide sequence and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0049] The vector containing the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells or recipient cells.

[0050] The present invention also provides a method for gene editing, including gene editing mediated by the cytosine base editor described herein. In addition to using the cytosine base editor described herein for gene editing, other gene editing reagents can be used as known in the art, for example, sgRNA can be designed using methods known in the art.

[0051] In the present invention, there is no particular limitation on the objects of gene editing, which can be somatic cells or germ cells, animal cells or human cells.

[0052] The present invention expands the application of CBE in laboratory and clinic.

[0053] Finally, it should be noted that the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art may make various corresponding changes and modifications based on the present invention, but these corresponding changes and modifications should all fall within the scope of protection of the claims attached to the present invention.

Claims

1. A cytosine deaminase, characterized in that: The cytosine deaminase is named SvSddA, and its amino acid sequence is shown in SEQ ID NO.

1.

2. A polynucleotide sequence characterized by: The nucleotide sequence thereof is shown in SEQ ID NO. 2, and is used for expressing the cytosine deaminase as claimed in claim 1.

3. A fusion protein, characterized in that: It comprises a Cas enzyme domain, a cytosine deaminase domain and a UGI domain; the cytosine deaminase domain is the cytosine deaminase as claimed in claim 1, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.

3.

4. A polynucleotide sequence characterized by: Its nucleotide sequence is shown in SEQ ID NO. 4, and it is used to express the fusion protein as claimed in claim 3.

5. A recombinant expression vector, characterized in that: It comprises the polynucleotide sequence according to claim 4.

6. A host cell, characterized in that: The invention comprises the recombinant expression vector according to claim 5 or the genome of which is integrated with the polynucleotide sequence according to claim 4, sgRNA, and AAV.

7. A cytosine single-base editor, characterized in that The cytosine single-base editor is obtained by integrating the polynucleotide sequence as described in claim 4 into an expression vector.

8. Use of the cytosine base editor according to claim 7 in preparing a reagent for mediating gene editing, characterized in that Including use in gene editing to reduce off-target effects, improve targeted editing efficiency or improve the fidelity of targeted editing.

9. A gene editing method, characterized in that: It includes mediating gene editing with the cytosine base editor as described in claim 7, specifically injecting the polynucleotide sequence as described in claim 4 and sgRNA into the host cell together.

10. A reagent or kit for gene editing, characterized in that: Including the cytosine deaminase as described in claim 1 or the cytosine base editor as described in claim 7.

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