Mutants of nuclease TnpB, their preparation methods and applications

Abstract

This invention discloses mutants of the nuclease TnpB, their preparation methods, and applications. Addressing the low editing efficiency of TnpB, this invention mutated TnpB to obtain five mutants, whose amino acid sequences are shown in SEQ ID Nos. 92-96. The editing efficiency of TnpB and its five mutants was tested by targeting four genes. The results showed that TnpB and its mutants TA, TC, and TD all exhibited high editing efficiency. While maintaining high editing efficiency, TnpB and the TD mutants showed low off-target rates at these sites. The TD mutant exhibited significantly more editing types than TnpB, and its ability to edit long fragments among the four targets was superior to TnpB. The TnpB mutants provided by this invention have higher gene editing efficiency and more editing types, showing promising applications in gene editing.

Description

Technical Field

[0001] This invention relates to enzyme mutants, and more particularly to mutants of the nuclease TnpB, their preparation methods, and their applications in gene editing, belonging to the field of nuclease mutants and their applications. Background Technology

[0002] The rapid development of gene editing technology has revolutionized life science research, with the CRISPR-Cas system becoming a mainstream tool due to its simplicity and high editing efficiency. However, the large molecular weight and limited delivery efficiency of Cas proteins still restrict their widespread application.

[0003] TnpB is an RNA-guided nuclease derived from the IS200 / IS605 transposon family in prokaryotes. It is considered the ancestor of the Cas12 protein and has a smaller protein size and simpler system structure, thus showing unique advantages in delivery and packaging. However, its editing efficiency is still relatively low and needs to be modified to improve its editing efficiency. Summary of the Invention

[0004] One objective of this invention is to provide a mutant of the nuclease TnpB;

[0005] A second objective of this invention is to provide the encoding gene of the mutant of the nuclease TnpB;

[0006] A third objective of this invention is to provide an expression cassette containing the coding gene of the mutant, a recombinant expression vector, or a recombinant host cell containing the recombinant expression vector;

[0007] The fourth objective of this invention is to apply the mutant of the nuclease TnpB, its encoding gene, an expression cassette or recombinant expression vector containing the encoding gene of the mutant, or a recombinant host cell containing the recombinant expression vector to gene editing.

[0008] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0009] One aspect of the present invention provides a mutant of the nuclease TnpB, wherein the amino acid sequence of the mutant is selected from any one of the amino acid sequences shown in SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. 95, or SEQ ID No. 96; preferably, the amino acid sequence of the mutant is selected from any one of the amino acid sequences shown in SEQ ID No. 93 or SEQ ID No. 95; most preferably, the amino acid sequence of the mutant is shown in SEQ ID No. 95.

[0010] The amino acid sequence of the nuclease TnpB described in this invention is shown in SEQ ID No. 1.

[0011] Another aspect of the present invention is to provide the coding gene for a mutant of the nuclease TnpB.

[0012] Another aspect of the present invention is to provide an expression cassette or recombinant expression vector containing the coding gene of a mutant of the nuclease TnpB, or a recombinant host cell containing the recombinant expression vector; wherein the recombinant expression vector may be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

[0013] The present invention further provides a method for preparing a mutant of any of the nucleases TnpB, comprising:

[0014] (1) The coding gene of the mutant nuclease TnpB is operatively linked with an expression regulatory element to construct a recombinant expression vector;

[0015] (2) Transform the recombinant expression vector into host cells, culture the host cells, induce the expression of recombinant protein, and purify the crude protein obtained.

[0016] Another aspect of the present invention is to apply the mutant of the nuclease TnpB, its encoding gene, an expression cassette or recombinant expression vector containing the encoding gene of the unit point mutant, or a recombinant host cell containing the recombinant expression vector to gene editing.

[0017] In a preferred embodiment of the present invention, the present invention provides a method for applying a mutant of the nuclease TnpB to gene editing, comprising: applying the mutant of the nuclease TnpB as the Cas protein in the CRISPR-Cas system to gene editing.

[0018] This invention addresses the issue of low editing efficiency when TnpB is used as a Cas protein. Five mutants were obtained by mutating the TnpB protein. The editing efficiency of wild-type TnpB and its five mutants was then tested by targeting four genes: AGBL, ROSA26, EMX1, and AAVS4. Sequencing results showed that wild-type TnpB and its mutants TA, TC, and TD exhibited high editing efficiency at AGBL-1, AGBL-2, ROSA26-1, ROSA26-3, and AAVS2 sites. Compared to the wild-type, the TD mutant showed approximately 20%, 21%, 46%, and 50% higher editing efficiencies at AGBL-1, AGBL-2, ROSA26-1, and ROSA26-3 sites, respectively. Furthermore, while maintaining high editing efficiency, both wild-type TnpB and the TD mutant exhibited low off-target rates at these sites. The types of edits with an efficiency of over 1 / 1000 were statistically analyzed. Overall, the TD mutant showed significantly more edit types than the TnpB mutant, and its ability to edit long fragments at the four targets was superior to that of the wild-type TnpB. Compared to the wild-type TnpB, the TnpB mutant provided by this invention, as a Cas protein in the CRISPR-Cas system, has higher gene editing efficiency and more edit types, and as a tool enzyme in the CRISPR-Cas system, it has significant application prospects in gene editing.

[0019] Definitions of terms involved in this invention

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, apparatus, and materials are now described.

[0021] The terms "polynucleotide" or "nucleotide" refer to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, specific nucleic acid sequences implicitly encompass variants of their conserved modifications (including (but not limited to) degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues. Mol Cell Probes 8:91-98 (1994)).

[0022] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms cover amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0023] The terms “mutation” and “mutant” have their common meanings here, referring to genetic, naturally occurring or introduced changes in a nucleic acid or polypeptide sequence, and their meanings are the same as those commonly known to those skilled in the art.

[0024] The terms "recombinant host cell line" or "host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotides may be maintained as, for example, non-integrating vectors of plasmids or may be integrated into the host genome. The host cell may be a prokaryotic or eukaryotic cell.

[0025] The term "operable connection" refers to a functional connection between two or more elements, which can be adjacent or non-adjacent.

[0026] The term "transformation" refers to the genetic conversion of polynucleotides or polypeptides into host cells by introducing gene-coding molecules into the host cells.

[0027] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in host cells. Attached Figure Description

[0028] Figure 1 The results show the editing efficiency and off-target rate analysis of TnpB wild-type and 5 TnpB mutants; where A represents the editing efficiency analysis results of TnpB wild-type and 5 TnpB mutants, and B represents the off-target rate analysis results of TnpB wild-type and mutant TD.

[0029] Figure 2 The results of the editing type analysis for TnpB wild-type and mutant TD. Detailed Implementation

[0030] The present invention will be further described below with reference to specific experimental examples, and the advantages and features of the present invention will become clearer with the description. However, these experimental examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0031] Example 1: Design, construction, and evaluation of editing efficiency and off-target rate of TnpB protein mutants.

[0032] 1. Experimental Methods

[0033] 1.1 Design and Construction of TnpB Protein and ωRNA

[0034] The IsDra2TnpB (TnpB) and its generated sequence were optimized using human codons and then ligated into the pRZ123 vector. Age I and Eco Between the two restriction enzyme sites RⅠ. The reRNA used is referenced in the article "Hypercompact TnpB and truncated TnpB systems enable efficient genome editing in vitro and in vivo". Bbs The protein and RNA sequences were linked together to form ωRNA, as shown in Table 1. Both the protein and RNA sequences were synthesized by General Biosciences.

[0035] The amino acid sequence of the IsDra2TnpB protein is shown below:

[0036] MIRNKAFVVRLYPNAAQTELINRTLGSARFVYNHFLARRIAAYKESGKGLTYGQTSSELTLLKQAEETSWLSEVDKFALQNSLKNLETAYKNFFRTVKQSGKKVGFPRFRKKRTGESYRTQFTNNNIQIGEGRLKLPKLGWVKTKGQQDIQGKILNVTVRRIHEGHYEASVLCEVEIPYLPAAPKFAAGVD VGIKDFAIVTDGVRFKHEQNPKYYRSTLKRLRKAQQTLSRRKKGSARYGKAKTKLARIHKRIVNKRQDFLHKLTTSLVREYEIIGTEHLKPDNMRKNRRLALSISDAGWGEFIRQLEYKAAWYGRLVSKVSPYFPSSQLCHDCGFKNPEVKNLAVRTWTCPNCGETHDRDENAALNIRREALVAAGIS (SEQ ID No.1).

[0037] Table 1 Target Names and Sequence Information

[0038]

[0039]

[0040] 1.2 HEK293T cell transformation and culture

[0041] After resuscitating HEK293T cells, they were stably cultured in 10cm dishes for 2-3 passages before being passaged into 24-well plates, maintaining a cell density between 70% and 90%. 1 μg of plasmid and 2 μL of transfection reagent (Lipo293™ from Beyotime Biotechnology) were added to each well. Cells without plasmid transfection served as a control group. Three replicates were prepared for each group, and cells were harvested after 72 hours of culture.

[0042] 1.3 Editing efficiency and miss rate detection

[0043] Genomic DNA was extracted from the recovered cells and used as a PCR template. Primers were designed upstream and downstream of the target site and off-target sites to obtain PCR products of approximately 150 bp containing editing target or off-target sites, which were then subjected to NGS sequencing. Off-target sites were designed using the Cas-OFFinder tool, and the nucleotide sequences of the primers used are shown in Table 2.

[0044] Table 2 Primer names and sequences

[0045]

[0046] 2. Experimental Results

[0047] 2.1 Results of Editing Efficiency and Off-Target Rate Testing

[0048] Five TnpB mutants were selected: TA, TB, TC, TD, and TE. The amino acid sequences of these five mutants are as follows:

[0049] The amino acid sequence of mutant TA is as follows:

[0050] MIRNKAFVVRLYPNAAQTELINRTLGCARFVYNHFLARRIAAYKESGKGLTYGQTSSELTLLKQAEEYSWLSEVDKFALQNSLKNLETAYKNFFRTVKQSGKKVGFPRFRKKRTGQSYRTQFTNNNIQIGEGRLKLPKLGWVKTKGQQDIQGKILNVTVRRIHEGHYEASVLCEVEIPYLPAAPKFAAGVD LGIKDFAIVTDGVRFKHEQNPKYYRSTLKKLRKAQQTLSRRTKGSANYGKAKTKLARIHKRIVNKRQDFLHKLTTKLVREYEIIGTEHLKPDNMRKNRRLALSISDAGWGEFIRQLEYKAAWYGRLVSKVSPYFPSSQLCHDCGFKNPEVKDLSVRTWTCPNCGETHDRDENAALNIRREALVAAGIS (SEQ ID No.92).

[0051] The amino acid sequence of mutant TB is as follows:

[0052] MIRNKAFVVRLYPNAAQTNLINRTLGSARFVYNHFLARRIATYKETGKGLTYGQTSSQLTLLKQAEETAWLSEVDSFALQNSLKNLETAYKNFFRTVKQSGKKVGFPRFRKKRTGESYRTQNTNNNIQVGEGRLKLPKLGWVKTKGQQDIQGRILNVTVRRIHSGHYEASVLCEVEIPYLPAAPKFAAGVDLGIKDFAIVTDGVRFKHEQNPKYLRSTLKRLRKAQQTLSRRTKGSARYGKAKTKLARIHKRIVNKRQDFLHKLTTSLVREYEIIGTEHLKPDNMRKNRRLALSISDAGWGEFIRQLEYKAAWYGRLVVKVSPFFPSSQLCHDCGFKNPEVKNLAVRTWTCPNCGETHDRDENAALNIRRFALVAAGIS (SEQ ID No.93).

[0053] The amino acid sequence of mutant TC is as follows:

[0054] MIRNKAFVVRLYPNKAQTELINRTLGCARFVYNHFLARRIAAYKESGKGLTYGQTSSQLTLLKQTEETSWLSEVDKFALQNSLKNLETAYKNFFRTVKQSGKKVGFPRFRKKRTGESYRTQFTNNNIQIGEGRLKLPKLGWVKTKGQQDIQGKILNVTVRRIHEGHYEASVLCEVEIPYLPAAPKFAAGVDVGIKDFAIVTDGERFKHEQNPKYYRSTLKRLRKAQQTLSRRKKGSARYGKAKTKLARIHKRIVNKRQDFLHKLTTSLVREYEIIGIEHLKPDNMRKNRRLALSISDAGWGEFIRQLEYKAAWYGRLVSKVSPYFPSSQLCHCCGFKNPEVKNLALRTWTCPNCGETHDRDENAALNIRREALVAAGIS (SEQ ID No.94).

[0055] The amino acid sequence of mutant TD is as follows:

[0056] MIRNKAFVVRLYPNAAQTELINRTLGSARFVYNHFLARRIAFYKESGKGLTYGQTSSELTLLKQAEETSWLSEVDKFALQNSLKNLETAYKNFFRTVKQSGKKVGFPRFRKKRTGESYRTQFTNNNIQVGEGRLKLPKLGWVKTKGQQEIAGKILNVTVRRIHEGHYEASVLCEVEIPYLPAAPKFAAGVD LGIKDFAIVTDGVRFKHEQNPKYYRSTLKRLRKAQQTLSRRKKGSANYGKAKTKLARIHKRIVNQRQDFLHKLTTSLVREYEIIGTEHLKPDNMRKNRRLALSISDAGWGEFIRQLEYKAAWYGRLVSKVSPYFPSSQLCHDCGFKNPEVKNLAVRTWTCPNCGETHDRDENAALNIRREALVAAGIS (SEQ ID No.95).

[0057] The amino acid sequence of mutant TE is as follows:

[0058] MIRNKAFVVRLYPNAAQTELINRTLGSARFVYNHFLARRIAAYKESGKGLTYGQTSSQLTLLKQAEETSWLSEVDKFALQNSLKNLETAYKNFFRTVKQSGKKVGFPRFRKKRTGESYRTKFTNNNIQIGEGRLKLPKLGWVKTKGQRDIQGKILNVTVRRIHEGHYEASVLCEVEIPYLPAAPKFAAGVD VGIKDFAIVTDGVRFKHEQNPKYYRSTLKRLRKAQQTLSRRKKGSARYGKAKTKLARIHKRIRNKRKDFLHKLTTSLVREYEIIGTETLKPDNMRKNRKLALSISDAGWGEFIRQLEYKAAWYGRTVIKVSPYFPSSQLCHDCGFKNPEVKNLAVRTWTCPNCGETHDRDENAALNIRREALVAAGIS (SEQ ID No.96).

[0059] To verify the editing efficiency of the five selected TnpB mutants, they were optimized according to human codons and constructed into vectors, which were then transformed into HEK293T cells. The vectors were designed to target four genes: AGBL, ROSA26, EMX1, and AAVS, and eight target sites were designed to detect the editing efficiency.

[0060] Sequencing results showed that wild-type TnpB and its mutants TA, TC, and TD exhibited high editing efficiency at AGBL-1, AGBL-2, ROSA26-1, ROSA26-3, and AAVS2 sites. Notably, compared with wild-type, the TD mutant showed approximately 20%, 21%, 46%, and 50% higher editing efficiency at AGBL-1, AGBL-2, ROSA26-1, and ROSA26-3 sites, respectively. Figure 1 A). Furthermore, while maintaining efficient editing, both wild-type TnpB and TD mutants exhibited low off-target rates at these sites ( Figure 1 B).

[0061] 2.2 Results of Editing Type Analysis

[0062] Editing types with an efficiency of over 1 / 1000 were statistically analyzed. The results showed that the editing types differed across the four target sites: AGBL-1, AGBL-2, ROSA26-1, and ROSA26-3. In AGBL-1, TnpB and the mutant TD primarily caused deletions of 3 bases at positions 15-17; in AGBL-2, deletions of 4 bases at positions 15-18; in ROSA26-1, deletions of 2 bases at positions 18-19; and finally, in ROSA26-3, TnpB primarily caused deletions of 4 bases at positions 16-19 and 3 bases at positions 18-20, while the mutant TD primarily caused deletions of 12 bases at positions 17-28. Overall, the mutant TD exhibited significantly more editing types than TnpB, and its ability to edit long fragments was superior to that of the wild-type TnpB across all four targets. Figure 2 ).

Claims

1. Mutant of the nuclease TnpB, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID No.

95.

2. A coding gene of the mutant of claim 1.

3. An expression cassette or a recombinant expression vector containing the coding gene of claim 2.

4. A recombinant host cell containing the expression cassette or the recombinant expression vector of claim 3.

5. A method of preparing the mutant of claim 1, wherein, Comprise: (1) The coding gene of the mutant is operably linked to an expression regulatory element to construct a recombinant expression vector; (2) The recombinant expression vector is transformed into a host cell, the host cell is cultured, the expression of the recombinant protein is induced, and the obtained crude protein is purified, thereby obtaining the mutant.

6. The mutant of claim 1 is used in gene editing; the gene editing is not used for human or animal bodies, and is not used for diagnostic or therapeutic purposes.

7. The coding gene of claim 2, the expression cassette or the recombinant expression vector of claim 3, and the recombinant host cell of claim 4 are used in gene editing; the gene editing is not used for human or animal bodies, and is not used for diagnostic or therapeutic purposes.

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