Cas effector protein mutants and related biomaterials and applications

Abstract

The application discloses a Cas effector protein mutant in the field of variation or genetic engineering and related biological materials and application thereof. The technical problem to be solved by the application is how to efficiently edit plant nucleic acids or how to improve the gene editing efficiency of CRISPR-Cas effector protein. Experiments prove that the gene editing efficiency of the Casdelta mutant obtained by the application on the rice OsPi21 target point is 30.6%, the gene editing efficiency on the rice OsPDS target point is 2%, and the gene editing efficiency on the rice OsALS target point is 16%. Compared with the Casdelta effector protein before modification, the editing activity is enhanced on different rice target points. The Cas effector protein mutant can be widely applied to the research on gene functions of rice and other crops and breeding improvement.

Description

Technical Field

[0001] This invention pertains to Cas effector protein mutants and their related biomaterials and applications in the field of mutation or genetic engineering. Background Technology

[0002] While the traditional CRISPR-Cas9 system is widely used, its large molecular weight (usually >1000 amino acids) makes it difficult to perform multi-gene editing or achieve efficient plant genetic transformation using a single vector. In addition, Cas9's strict prototypical spacer adjacent motif (PAM) recognition bias limits its targeting flexibility across the genome.

[0003] To overcome the aforementioned limitations, researchers have recently focused on discovering and developing novel small Cas effector proteins, such as Casδ, Cas12f, and Cas14 subtypes derived from different microbial species. These novel Cas proteins possess significant structural advantages: firstly, their smaller molecular weight facilitates delivery to monocotyledonous and dicotyledonous plant cells via Agrobacterium-mediated transformation and also enables in vivo delivery of adeno-associated virus (AAV) vectors; secondly, they often recognize PAM sequences different from Cas9, enabling them to cover genomic sites inaccessible to the traditional Cas9 system. Therefore, developing and optimizing such novel Cas effector proteins is a crucial research direction in the field of gene editing. Against this backdrop, Casδ proteins, with their small protein conformation and non-classical PAM recognition mechanism, represent an important compact gene editing tool, potentially offering further solutions to the insufficient targeting flexibility of traditional CRISPR systems in complex plant genomes.

[0004] However, although these novel Cas effector proteins have shown activity in in vitro biochemical experiments or prokaryotic cells, their editing efficiency is often far lower than expected in actual plant cell environments. To address these issues, protein engineering techniques are commonly used to optimize Casδ proteins. Commonly used protein optimization methods mainly include directed evolution and structure-guided rational design. However, these methods suffer from drawbacks in practical applications, such as low efficiency, high cost, and strong dependence on structural information.

[0005] In summary, there is a need in the existing technology for an improved scheme that does not rely on complex structural information and can significantly improve the editing efficiency of novel Cas effector proteins (especially Casδ) in plant cells, so as to meet the actual needs of precision crop breeding and the development of plant synthetic biology. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to efficiently edit plant nucleic acids or how to improve the editing efficiency of CRISPR-Cas effector protein Casδ (amino acid sequence is sequence 1 in the sequence listing).

[0007] To address the aforementioned technical problems, this invention first provides a Cas effector protein mutant. The Cas effector protein mutant is Casδ-N915L or a fusion protein containing Casδ-N915L. Casδ-N915L can be a protein whose amino acid sequence is sequence 3 in the sequence listing. The fusion protein is Casδ-N915L-NLS, and Casδ-N915L-NLS can be a protein whose amino acid sequence is sequence 6 in the sequence listing.

[0008] The amino acid residues 1-936 of sequence 6 are the Casδ-N915L protein sequence, the amino acid residues 937-940 are the Linker sequence, and the amino acid residues 941-956 are the Os-N-NLS nuclear localization signal sequence.

[0009] The Cas effector protein mutants mentioned above can be proteins with RNA-directed DNA endonuclease activity.

[0010] The Cas effector protein mutants mentioned above can be CRISPR-Cas RNA-guided peptides. These mutants cleave nucleic acids under the guidance of the CRISPR / Cas RNA, thereby enabling the editing of the nucleic acids to be edited.

[0011] Cas refers to CRISPR-associated protein. In one specific embodiment of the present invention, the Cas is the Casδ protein.

[0012] To address the aforementioned technical problems, the present invention also provides a nucleic acid molecule. The nucleic acid molecule may be the encoding gene of the Cas effector protein mutant. The encoding gene may be the Casδ-N915L gene or the Casδ-N915L-NLS gene.

[0013] The nucleotide sequence of the Casδ-N915L gene may be sequence 2 in the sequence listing. The coding sequence of the Casδ-N915L-NLS gene may be sequence 7 in the sequence listing.

[0014] In some embodiments of the present invention, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0015] The nucleic acid molecule described above encodes the Cas effector protein mutant described above.

[0016] To address the aforementioned technical problems, the present invention also provides an expression cassette. The expression cassette may contain the nucleic acid molecules described above.

[0017] The expression cassette described above is a DNA molecule capable of expressing the coding gene of the Cas effector protein mutant in a host cell (such as a plant cell). In some embodiments, the expression cassette further includes a DNA fragment containing all the regulatory sequences necessary for expressing the coding gene. The regulatory sequences, under compatible conditions, guide the expression of the coding gene in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences include a promoter and termination signals for transcription and translation. The regulatory sequence can be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to that of the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence is operatively linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that functions in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that functions in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell used can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby opening or closing gene expression. Other examples of regulatory sequences are those that enable gene amplification. In these examples, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0018] In one embodiment, the expression cassette is expression cassette 1 of the Casδ mutant Casδ-N915L, and the nucleotide sequence of the expression cassette is sequence 5 in the sequence listing.

[0019] To address the aforementioned technical problems, the present invention also provides a recombinant vector. The recombinant vector may contain the nucleic acid molecules described above.

[0020] The term "vector" refers to any construct that can introduce heterologous DNA or RNA into a host cell.

[0021] The term "construct" refers to any recombinant DNA or recombinant RNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, or bacteriophages. Constructs typically include one or more expression cassettes.

[0022] In some embodiments, the recombinant vector is a recombinant expression vector containing the coding gene of the Cas effector protein mutant. When preparing the recombinant expression vector, the coding gene may be located within the vector so as to be operatively linked to an appropriate expression regulatory sequence. The recombinant expression vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA manipulation and expression of the coding gene. The choice of vector typically depends on its compatibility with the host cell into which it will be introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector (i.e., a complete structure existing outside the chromosome that can replicate independently of the chromosome), such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector may contain any mechanism that ensures self-replication. Alternatively, the vector is one that, when introduced into a host cell, will integrate into the genome and replicate along with the integrated chromosome.

[0023] In this application, the term "operable link" refers to an operable connection between segments in a nucleic acid sequence that are functionally related to each other. For example, operably linked promoters, enhancers, open reading frames, 5'UTRs and 3'UTRs, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements lead to transcription of the open reading frame and ultimately to the production of a polypeptide (i.e., expression of the open reading frame).

[0024] In one embodiment, the recombinant vector is a step3 recombinant vector, and the nucleotide sequence of the recombinant vector is sequence 39 in the sequence listing.

[0025] To address the aforementioned technical problems, the present invention also provides recombinant microorganisms. These recombinant microorganisms may contain the Cas effector protein mutants or nucleic acid molecules described above, and are capable of expressing proteins with RNA-directed DNA endonuclease activity.

[0026] To address the aforementioned technical problems, this invention also provides the application of a protein in rice nucleic acid editing, wherein the protein is a mutant of the Cas effector protein described above.

[0027] In some embodiments, the nucleic acid editing is DNA editing.

[0028] In some embodiments, the DNA editing is gene editing.

[0029] In one specific embodiment of the present invention, the gene may be from the rice genome. OsPDS, OsALS and OsPi21 Genes. In one specific embodiment of the present invention, the recombinant microorganism is recombinant Escherichia coli and / or recombinant Agrobacterium.

[0030] To address the aforementioned technical problems, the present invention also provides a method for editing rice nucleic acids, the method comprising contacting a guide RNA targeting the nucleic acid to be edited and the Cas effector protein mutant described above with the rice genome to achieve nucleic acid editing of the rice genome.

[0031] The contact can occur within a cell, and the nucleic acid editing can be DNA editing.

[0032] This invention belongs to the fields of biotechnology and genetic engineering. It provides a highly active Casδ mutant, Casδ-N915L, and its application in plant genome editing, aiming to overcome the low editing efficiency of existing Casδ systems in plants and provide a superior gene editing tool for plant biotechnology and crop breeding.

[0033] The Casδ mutant Casδ-N915L obtained in this invention exhibits significantly enhanced genome editing efficiency in rice. Results in the examples show that Casδ-N915L achieves approximately 30.6% gene editing efficiency at the OsPi21 target in rice, approximately 2% at the OsPDS target, and approximately 16% at the OsALS target. Compared to the unmodified Casδ effector protein (editing efficiency of 1% at the OsPi21 target, no editing activity detected at the OsPDS target, and 10% at the OsALS target), it demonstrates enhanced editing activity at various rice targets. The Casδ-N915L of this invention can be widely applied to gene function research and breeding improvement in rice and other crops. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the vector structure for co-expressing sgRNA expression cassettes and Casδ mutant expression cassettes targeting different genes. The Casδ protein variant represents Casδ-N915L.

[0035] Figure 2 This study compares the editing efficiency of wild-type Casδ protein (WT) and the mutant effector protein Casδ-N915L. Efficiency is expressed as the percentage of T0 generation plants with mutations at the target site. The vertical axis represents editing efficiency (mutation rate), expressed as a percentage, calculated as "number of T0 generation plants with mutations at the target site / total number of T0 generation gene-edited plants tested × 100%". The horizontal axis represents wild-type Casδ protein (WT) and the mutant effector protein Casδ-N915L. A represents the editing efficiency of wild-type Casδ protein (WT) and the mutant effector protein Casδ-N915L in the rice genome. OsALSComparison of gene editing efficiency; B represents the efficiency of gene editing in the rice genome. OsPDS Comparison of gene editing efficiency; C represents the efficiency of gene editing in the rice genome. OsPi21 Gene editing efficiency was compared. The fractions on the bars in the graph represent gene editing efficiency, with the numerator being the number of mutant strains and the denominator being the total number of strains tested. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] The WMC025 vector in this embodiment of the invention is derived from Weimi Biotechnology Co., Ltd.

[0039] Example 1. Optimization design of CRISPR-Cas effector proteins based on artificial intelligence (AI) models.

[0040] 1. Mutation recommendation and design based on protein language model (ESM).

[0041] This invention inputs the protein sequence (Sequence 1 in the sequence listing) of wild-type Casδ (from Professor Lai Jinsheng's laboratory at China Agricultural University) into a protein language model to screen for candidate amino acid mutation sites.

[0042] 1.1 Model and Input.

[0043] Six large masked protein language models (including one ESM-1b and five ESM-1v series models, all of which are open source models) were used, with wild-type (i.e. unmodified) Casδ protein sequences as the sole input.

[0044] Sequence 1: 。

[0045] 1.1.1 Download pre-training model。

[0046] Download one ESM-1b (esm1b_t33_650M_UR50S) and five ESM-1v (esm1v_t33_650M_UR90S_1, esm1v_t33_650M_UR90S_2, esm1v_t33_650M_UR90S_3, esm1v_t33_650M_UR90S_4, esm1v_t33_650M_UR90S_5) pre-trained models, including model weights and configuration files, from the GitHub repository (https: / / github.com / facebookresearch / esm).

[0047] 1.1.2 Loading the model.

[0048] Using a Python programming environment, the esm package installed from the GitHub repository https: / / github.com / facebookresearch / esm is used to call the loading functions provided by its esm.pretrained module (including esm.pretrained.esm1b_t33_650M_UR50S() and esm.pretrained.esm1v_t33_650M_UR90S_1()) to load the model weights and corresponding amino acid alphabets of the pre-trained ESM-1b and ESM-1v models.

[0049] 1.1.3 Input protein sequences into the model to generate embeddings.

[0050] The protein sequence (Casδ protein sequence, sequence 1 in the sequence listing) can be directly input into the model loaded in step 1.1.2 without any additional transformation, and the embedded information vector corresponding to the Casδ protein sequence will be output.

[0051] 1.2 Mutation calculation and screening.

[0052] For each amino acid site in the Casδ protein sequence, the model calculates the likelihood score for all 20 possible amino acid substitutions. This invention calculates the "log-likelihood ratio" of each amino acid substitution relative to the wild-type amino acid. Only mutations whose likelihood scores are higher than those of the wild-type amino acid ("log-likelihood ratio" greater than 0) in multiple model consensus are selected as candidates. Furthermore, by setting a "log-likelihood ratio" threshold, this invention filters out a small number (<20) of high-potential candidate point mutations from all candidate mutations for low-throughput experimental validation.

[0053] 1.3 Candidate mutations.

[0054] Ultimately, this invention selected candidate single-point amino acid substitution mutations based on a threshold (i.e., a log-likelihood ratio greater than 0) for in vivo functional verification.

[0055] 2. Rational arginine mutation design based on three-dimensional structure prediction.

[0056] 2.1 Structural prediction.

[0057] First, the three-dimensional structure of the Casδ protein was predicted using the AlphaFold3 online tool (https: / / alphafoldserver.com / ).

[0058] 2.2 Identification of key residues.

[0059] Given that Casδ has a highly similar protein structure to the resolved Cas12h1 (PDB: 8Y9N) (RMSD=4.576), this invention uses the Cas12h1 structure (https: / / www.rcsb.org / structure / 8Y9L) as a template and uses the distance function built into the PYMOL software (parameter settings mode=polar, cutoff=4.0) to identify the amino acid residues that interact with crRNA or DNA substrates.

[0060] 2.3 Homologous mapping and design.

[0061] By using structural alignment based on the amino acid correspondences of protein structures provided by PYMOL, the nucleic acid interaction amino acid residue sites identified in Cas12h1 are mapped to the predicted Casδ structure, thus obtaining amino acid residue sites in the Casδ protein sequence that have potential interaction capabilities with crRNA and substrate DNA. Subsequently, this invention selects candidate residue sites with non-positive charges and mutates them to positively charged arginine (R) residues, aiming to increase electrostatic interactions with the negatively charged nucleic acid backbone, thereby improving editing efficiency.

[0062] Based on the two strategies in the above steps, the present invention screens all designed Casδ mutants to obtain the Casδ mutant Casδ-N915L. This mutant is a mutant protein obtained by mutating the 915th amino acid residue of sequence 1 in the sequence listing from N (asparagine) to L (leucine). The amino acid sequence of Casδ-N915L is shown in sequence 3 in the sequence listing.

[0063] Casδ-N915L protein sequence (Sequence 3): 。

[0064] Example 2. Optimizing the construction of the expression vector of the protein Casδ-N915L effect mutation.

[0065] 1. Construction of the sgRNA expression vector for the gene to be edited.

[0066] 1.1 Design of sgRNA for the gene to be edited and synthesis of sgRNA tandem expression cassette.

[0067] To evaluate the gene editing efficiency of the Casδ mutant Casδ-N915L, this invention designs genes that target endogenous genes in rice. OsALS, OsPDS and OsPi21 Three crRNA expression cassettes targeting different targets were designed, and the expression cassette of the Casδ mutant (Casδ-N915L) obtained after optimization in Example 1 was constructed on the same vector.

[0068] The design of the single guide RNA target sequence is based on three rice genes. OsALS, OsPDS and OsPi2 The design of genome sequences, specifically targeting OsALS The nucleotide sequence of the target sequence named sgRNA1 is 5' ATCCCAAGTGGGGGCGCATTCAAGGA-3' (Sequence 8), and it targets the sequence shown in Sequence 40 of the sequence listing. OsALS Nucleotides 1920-1945 of the gene; targeting OsPDS The nucleotide sequence of the target sequence named sgRNA2 is 5'-AACATAACTGGAACCAGCCAAGCAAG-3' (Sequence 9), and it targets the sequence shown in Sequence 41 of the sequence listing. OsPDS Nucleotides 28-53 of the gene; targeting OsPi21 The nucleotide sequence of the target sequence named sgRNA3 is 5'-GGTATATTGGTCATCTTGGTGGACCT-3' (Sequence 10), which targets the sequence shown in Sequence 42 of the sequence listing. OsPi21 Nucleotides 459-484 of the gene.

[0069] The sgRNA sequences of the three genes are respectively composed of OsU3 , OsU6a and OsU6b Promoter-driven tandem expression. A tandem expression cassette 2 (sequence 4 in the sequence listing) of three artificially synthesized sgRNA sequences of genes.

[0070] Sequence 4 (5'-3'):

[0071] 1.2 Enzyme digestion of sgRNA tandem expression cassette.

[0072] By means of PCR, BsaI and SbfI restriction sites were introduced at the 5' and 3' ends of the tandem expression cassette 2 of the three gene sgRNA sequences synthesized in step 1.1, respectively, for subsequent vector ligation. The PCR amplification program was as follows: 95℃, 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 30 cycles; 72℃, 5 min; stored at 4℃. The primers used for PCR were y2592230hyg-OSU31-F2-BsaI (sequence 36: 5'-atGGTCTCTtgtgtGTGCTGACGACCAGCACTAGATGGTCGTTCAGGCACAACATAACT-3') and y2592230hyg-OSU62-R2-SbfI (sequence 37: 5'-cacctgcaggaaacaaaaaaAGGTCCACCAAGATGACCAATATACCGTGCCTGAACGA-3').

[0073] After the above PCR amplification was completed, the PCR amplification product was digested with BsaⅠ and SbfI enzymes (37 ℃, 30 min) to obtain enzyme digestion product 1. Enzyme digestion product 1 was then recovered and purified (Novizan DC301 kit) to obtain sgRNA tandem expression cassette enzyme digestion product.

[0074] Enzyme digestion system (50 µL): 6 μL (1 μg) PCR product; 1 µL BsaⅠ enzyme; 1 µL SbfI enzyme; 5 µL Buffer; ultrapure water to bring the total to 50 µL.

[0075] 1.3 Linearization of expression vectors.

[0076] The WMC025 vector was digested using the following enzyme digestion system (37 °C, 30 min). The digestion products were purified and recovered (Novazia DC301 kit) to obtain a linearized vector, which was used to ligate the sgRNA tandem expression cassette digestion products.

[0077] Enzyme digestion system (50 µL): 6 μL (1 μg) WMC025 vector; 1 µL BsaⅠ enzyme; 1 µL SbfI enzyme; 5 µL buffer; ultrapure water to bring the total to 50 µL.

[0078] 1.4 Ligation and transformation to obtain the step 1 recombinant vector.

[0079] The linearized vector obtained in step 1.3 was ligated with the sgRNA tandem expression cassette digestion product obtained in step 1.2 to obtain ligation product 1. The ligation conditions were: ligation at 25 ℃ for 30 min, followed by storage on ice. The ligation system consisted of: T4 ligase, 1 µL; T4 ligase buffer, 1 µL; linearized vector, 30 ng; sgRNA tandem expression cassette digestion product, 3 µL; and ultrapure water to a final volume of 10 µL.

[0080] Take 5 µL of the above ligation product 1, add 50 µL of competent cells (DH5α), mix, and incubate on ice for 25 min (do not shake); gently remove, heat shock at 42 ℃ for 35 s, and immediately place on ice for 2 min; add 100 µL of LB medium, and incubate at 37 ℃ with shaking for 1 h; take 60 µL of bacterial culture and spread it on an LB agar plate containing kanamycin, and incubate overnight at 37 ℃ upside down. The next day, pick colonies for sequencing, sequencing primer (sequence 11): 5'-TCAAACAAGTGTGACAAAAA-3'.

[0081] Select a single positive colony that has been successfully sequenced (a recombinant vector that has successfully ligated the sgRNA tandem expression cassette) and shake it to extract plasmids to obtain the step 1 recombinant vector.

[0082] 2. Step 1: Recombinant vector to replace promoter and terminator.

[0083] The recombinant vector obtained in step 1.4 was modified by replacing the promoter with the UBI promoter and the Os-N-NLS promoter to insert the Casδ variant, and the terminator was replaced with the E9 terminator to obtain a higher expression level of the Casδ mutant effector protein.

[0084] 2.1 Enzyme digestion and recovery of the vector.

[0085] The step1 recombinant vector obtained in step 1.4 was digested with SbfI and EcoR1. After digestion at 37 °C for 30 min, the large fragment was recovered to obtain the linear step1 recombinant vector.

[0086] Enzyme digestion system: Step 1 recombinant vector, 1 μg (6 μL); SbfI enzyme, 1 µL; EcoRI enzyme, 1 µL; Buffer, 5 µL; Ultrapure water to bring the total to 50 µL.

[0087] 2.2 PCR amplification: PCR product 1 was obtained by amplifying the UBI promoter (template from plasmid WMC025, nucleotides 27-2011 of sequence 12) using UBI-CZF (sequence 13: 5'-tgcttttttgttttagagcctgcaggtg-3') and UBI-CZR (sequence 14: 5'-GCCTAGGgctgcagaagtaacaccaaacaacag-3') for subsequent experiments. NLS-CZF (sequence 15: 5'-acttctgcagcCCTAGGCCTACATGGGGCC-3') and NLS-CZR (sequence 16: 5'-GGTACCcaatcaggatccCTTGTCGTCATCATCTTTATAA) was used. PCR product 2 was obtained by amplifying the nuclear localization signal sequence Os-N-NLS (template from plasmid Os-N-NLS, Weimi Biotechnology Co., Ltd., nucleotides 2138-2185 of sequence 12) required for subsequent experiments using TCG-3'; PCR product 3 was obtained by amplifying the E9 terminator (template from plasmid HM-Os-new, Weimi Biotechnology Co., Ltd., nucleotides 2195-2829 of sequence 12) using E9-CZF (sequence 17: 5'-ggatcctgattgGGTACCAAAAGACCCGCAGCGACCAA-3') and E9-CZR (sequence 18: 5'-atgacatgattacgaattcGTTGTCAATCAATTGGCAAGTCAT-3') required for subsequent experiments using TCG-3'. The three PCR products were mixed as templates for a fourth PCR amplification (using UBI-CZF and E9-CZR primers) to link the three products into one fragment (UBI+Os-N-NLS+E9 terminator, sequence 12 in the sequence listing), resulting in PCR product 4. A KpnI restriction site was added between the Os-N-NLS and E9 terminators for subsequent experiments.

[0088] Sequence 12 (5'-3'):

[0089] 2.3 Connection and Transformation: The linear step1 recombinant vector obtained in step 2.1 was ligated with PCR product 4 obtained in step 2.2 to obtain ligation product 2. Ligation conditions: 50 ℃, ligation for 20 min, and storage on ice.

[0090] Ligation system: Recombinant ligase (TSINGKE TSV-S2 Trelief® SoSoo Cloning Kit Ver.2), 1 µL; linear step 1 recombinant vector, 30 ng; PCR product, 4 µL; ultrapure water to 10 µL.

[0091] Take 5 µL of the above ligation product 2 and transform it into E. coli DH5α competent cells. Incubate overnight at 37 ℃ with the cells inverted. The next day, pick colonies for sequencing. Extract plasmids from successfully sequenced single colonies using shaking culture to obtain the step 2 recombinant vector. Sequencing primers are as follows: E9-JJR (sequence 19): 5'-ACTCAGTAGGATTCTGGTGTGTG-3'; PCAMBIA1301-F (Sequence 20): 5'-CCAGGCTTTACACTTTATGC-3'; UBI-SEQR (sequence 21): 5'-TCCGCCACCGCAATTTCTGGA-3'; UBI-SEQ1 (Sequence 22): 5'-AACGGACACCAACCAGCGAACCAG-3'.

[0092] 3. Construction and vector ligation of the Casδ mutant Casδ-N915L expression cassette.

[0093] 3.1 Synthesis of the gene encoding the Casδ mutant Casδ-N915L.

[0094] The Casδ mutant Casδ-N915L was codon-optimized (for rice) and its coding sequence (Sequence 2 in the sequence listing, corresponding to nucleotides 2094-4901 of Sequence 5 in the sequence listing) was synthesized (Nanjing Genscript Biotech Co., Ltd.).

[0095] In step 3.2, the codon-optimized Casδ-N915L coding sequence is constructed into the recombinant vector obtained in step 2, resulting in a co-expression vector of Casδ-N915L and sgRNA. In this co-expression vector, Casδ-N915L is derived from maize ubiquitin protein (…). ZmUBI The promoter drives expression, forming the expression cassette 1 of Casδ-N915L. Figure 1 The expression box 1 sequence corresponds to sequence 5 in the sequence list.

[0096] Sequence 2 (5'-3'):

[0097] 3.2 Obtaining the Casδ-N915L and sgRNA co-expression vector.

[0098] 3.2.1 Step 2: Linearization of the recombinant vector.

[0099] The Step 2 recombinant vector obtained in step 2 was digested using the following enzyme digestion system and then purified and recovered to obtain the linearized Step 2 recombinant vector. Enzyme digestion conditions: 37 ℃, 30 min.

[0100] Enzyme digestion system: Step 2 recombinant vector, 1 μg (6 μL); KpnI enzyme, 1 µL; Buffer, 5 µL; Ultrapure water to bring the total to 50 µL.

[0101] 3.2.2 Amplify the DNA sequence of the Casδ variant Casδ-N915L.

[0102] PCR amplification was performed using the CDS of the Casδ variant Casδ-N915L shown in sequence 2 as a template to obtain PCR product 5, which is the Casδ-N915L CDS sequence with added recombination arms. The primers used were Cas-CZF (sequence 23: 5'-TAAAGATGATGACGACAAGATGTCGAAAGACGGGGACAAG-3') and Cas-CZR (sequence 24: 5'-TGGTCGCTGCGGGTCTTTTGGACCCGGAACCAACAACACT-3').

[0103] 3.2.3 Connection and transformation.

[0104] The PCR product containing the nucleotide sequence of the Casδ variant Casδ-N915L (PCR product 5) was ligated into the step2 recombinant vector to obtain ligation product 3, which contains tandem expression cassette 2 with three sgRNA targets and expression cassette 1 with the Casδ variant Casδ-N915L. This ligation product was used for subsequent experiments. Ligation system: Recombinant ligase (TSINGKE TSV-S2 Trelief® SoSoo Cloning Kit Ver. 2), 1 µL; linearized step2 recombinant vector, 30 ng; PCR product 5, 3 µL; ultrapure water to a final volume of 10 µL.

[0105] Ligation reaction conditions: 50 ℃, ligation for 20 min, then stored on ice.

[0106] Take 5 µL of the above ligation product 3 and transform it into E. coli DH5α competent cells, and incubate overnight at 37 ℃ inverted position. The next day, pick colonies for sequencing. Extract plasmids from successfully sequenced single colonies by shaking, yielding a step3 recombinant vector containing a tandem expression cassette 2 of three sgRNA targets and an L expression cassette 1 of the Casδ variant Casδ-N915. The nucleotide sequence of the step3 recombinant vector is sequence 39 in the sequence listing. Nucleotides 274-1674 of sequence 39 correspond to the tandem expression cassette 2 of the three gene sgRNA sequences (sequence 4 in the sequence listing), and nucleotides 1694-7298 of sequence 39 correspond to the expression cassette 1 of Casδ-N915L (sequence 5 in the sequence listing).

[0107] Sequencing primers: ubi-seqF (sequence 25): 5'-CAGCAGCTATATGTGGATTT-3'; E9-JJR (sequence 26): 5'-ACTCAGTAGGATTCTGGTGTGTG-3'; Cas-seqR1 (sequence 27): 5'-ATGCCAGGCGTCTTCTTGCT-3'; Cas-seqR2 (sequence 28): 5'-ACAGATGATCACCGGCTCGC-3'; Cas-seqR3 (sequence 29): 5'-ATGACGGGGAGGTAATGTGA-3'.

[0108] In cassette 1 of Casδ-N915L, nucleotides 1-1985 of sequence 5 are the UBI promoter sequence; the coding sequence of Casδ-N915L (sequence 2) corresponds to nucleotides 2094-4901 of sequence 5; nucleotides 4902-4913 of sequence 5 are the linker amino acid coding sequence; nucleotides 4914-4961 of sequence 5 are the Os-N-NLS nuclear localization signal coding sequence; and nucleotides 4971-5605 of sequence 5 are the E9 terminator sequence.

[0109] The Casδ-N915L expression cassette 1 expresses the Casδ-N915L protein linked to the NLS nuclear localization signal, named Casδ-N915L-NLS. The amino acid sequence of Casδ-N915L-NLS is sequence 6 in the sequence listing, and its encoding nucleotide sequence is sequence 7 in the sequence listing. Amino acid residues 1-936 of sequence 6 constitute the Casδ-N915L protein sequence, amino acid residues 937-940 constitute the linker sequence, and amino acid residues 941-956 constitute the Os-N-NLS nuclear localization signal sequence.

[0110] Sequence 5 (5'-3'):

[0111] sequence6: 。

[0112] Sequence 7 (5'-3'):

[0113] Example 3. Validation of gene editing efficiency of the Casδ mutant Casδ-N915L.

[0114] 1. Obtaining recombinant Agrobacterium.

[0115] The recombinant vector obtained in Example 2 (step3) was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / step3. The specific steps are as follows: Strain activation: Take out Agrobacterium strain (EHA105) from the -80℃ freezer, streak it onto a solid YEB plate containing rifampicin, and incubate it upside down at 28℃ for about 2 days until a single colony grows.

[0116] Liquid culture: Pick a single colony and inoculate it into 5 mL of liquid culture medium (YEB), and culture overnight at 28°C and 200 rpm with shaking to obtain the seed culture.

[0117] Expanding the culture: Transfer the seed culture to fresh culture medium at a ratio of 1:100, continue incubation at 28°C with shaking, and monitor the bacterial concentration continuously until the OD (Organic Degradation) is reached. 600nm Stop immediately when the value reaches 0.6, at which point the bacteria are in the logarithmic growth phase and are most likely to absorb exogenous DNA.

[0118] Competent cell treatment: The bacterial culture was placed in an ice bath for 30 minutes, centrifuged at 4°C to collect the cells, gently resuspended in pre-cooled CaCl2 solution (containing 15% glycerol), aliquoted, and flash-frozen in liquid nitrogen at -70°C for later use to obtain Agrobacterium competent cells.

[0119] Mixed plasmid: Take 100 μL of Agrobacterium competent cells and thaw them on ice. Add 1 μg of the recombinant vector plasmid DNA obtained in Example 2 (step3) to obtain a mixed solution. After gently mixing, incubate on ice for 30 minutes to allow the DNA to adsorb onto the cell surface.

[0120] Freeze-thaw transformation: The mixture is placed in liquid nitrogen for 5 minutes, then quickly transferred to a 37°C water bath for 5 minutes of heat shock, and then immediately placed in an ice bath for 5 minutes. The temperature difference changes create pores on the cell membrane.

[0121] Resuscitation culture: Add 800 μL of antibiotic-free YEB liquid medium and culture at 28°C and 200 rpm for 5 hours to obtain recombinant Agrobacterium EHA105 / step3.

[0122] Simultaneously, the step3-2 recombinant vector containing the wild-type Casδ sequence and three sgRNA target sequences was transformed into Agrobacterium competent cells to obtain recombinant Agrobacterium EHA105 / step3-2. The step3-2 recombinant vector was obtained by replacing the Casδ variant Casδ-N915L nucleotide sequence in the step3 recombinant vector with the wild-type Casδ coding sequence (sequence 38 in the sequence listing) while keeping other sequences unchanged. The construction method of the step3-2 recombinant vector was the same as that of the step3 recombinant vector in Example 2. Recombinant Agrobacterium EHA105 / step3-2 was used as a control for subsequent transformation into rice to compare and analyze the gene editing efficiency of the Casδ mutant Casδ-N915L.

[0123] 2. Rice transformation and the acquisition of gene-edited rice.

[0124] Recombinant Agrobacterium EHA105 / step3 and recombinant Agrobacterium EHA105 / step3-2 were introduced into embryogenic callus of the rice variety "Nipponbare" using Agrobacterium-mediated stable genetic transformation. After antibiotic screening and regeneration, T0 generation gene-edited plants were obtained.

[0125] 2.1 Callus induction and subculture.

[0126] Select mature rice seeds, remove the husks, pour them into 50mL centrifuge tubes, add 75% ethanol for 1 min to sterilize, discard the ethanol, rinse once with sterile water, discard the ethanol, then add 30% sodium hypochlorite for 20 min to sterilize, discard the sodium hypochlorite, and rinse 5 times with sterile water. Use a pipette to remove excess water (or use sterile filter paper to blot dry), and transfer the seeds to induction medium, 20 seeds per dish.

[0127] After callus grows, it can be directly transformed using the protoembryo. The small particles growing next to the protoembryo can be picked and placed on a new induction medium for subculture. When they grow to a suitable size, they can also be transformed.

[0128] 2.2 Agrobacterium culture.

[0129] Agrobacterium EHA105 containing the target gene vector was streaked on a plate containing the corresponding antibiotic (rifampin) and incubated in the dark at 28°C for 2 days until a single colony appeared.

[0130] 2.3 Agrobacterium infection.

[0131] Prepare the infection solution. Use a pipette to draw up the infection solution and wash off the recombinant Agrobacterium on the YEB plate to obtain the Agrobacterium suspension used for co-culture transformation of rice.

[0132] Select a sufficient number of callus tissues (good callus condition, bright yellow color, round and firm texture, and particle diameter of about 3 mm are preferred) and place them in a 100 mL sterile Erlenmeyer flask. Add an appropriate amount of Agrobacterium suspension (ensure sufficient bacterial suspension to contact the material). Incubate at room temperature for 20 minutes, shaking occasionally. Discard the bacterial suspension, place the callus tissues on sterile filter paper to absorb excess bacterial suspension, and then transfer them to a solid co-culture medium lined with a layer of sterile filter paper. Incubate at 26°C in the dark for 3 days.

[0133] 2.4 Screening and cultivation.

[0134] After 3 days of co-culture, the callus tissue needs to be cleaned. Using a 1mL blue pipette tip, transfer the callus from the co-culture medium to a sterile Erlenmeyer flask. Rinse twice with sterile water, and then rinse a third time with sterile water containing carbenicillin. After pipetting away excess water, transfer the callus to sterile filter paper and air dry it using the airflow of a laminar flow hood for approximately 30 minutes. Once dry, transfer the callus to selection medium for selection culture at 28°C in the dark. The selection process takes 3-4 weeks.

[0135] 2.5 Differentiation and regeneration.

[0136] One month after screening, bright yellow positive calluses were observed to grow. At this point, the positive calluses could be picked and transferred to differentiation medium for differentiation and regeneration. Sixteen positive calluses were placed on each differentiation dish and cultured under light at 28°C. After 10 days of culture, green spots appeared on the calluses, and after another 10 days of culture, T0 generation gene-edited plant seedlings were differentiated.

[0137] 2.6 Seedling rooting.

[0138] Once the differentiated seedlings have grown to about 2-3cm and have obvious roots, they can be transferred to a rooting medium to allow them to grow. The rooting medium should be poured into a relatively tall bottle or tube so that the rooted seedlings have enough space to grow tall. The rooting culture conditions are 28℃ and sterile light culture.

[0139] 3. Mutation detection and analysis of the target edited gene.

[0140] Genomic DNA was extracted from T0 generation gene-edited plants, and each target region was amplified by PCR and then subjected to Sanger sequencing (performed by Beijing Qingke Biotechnology Co., Ltd., Hainan Branch). The sequencing results were visualized using SnapGene software, and the editing efficiency (number of plants with target mutations / total number of plants tested) was calculated. A double peak at the target site indicates a gene-edited positive plant.

[0141] Table 1. Primers for gene editing efficiency detection.

[0142]

[0143] The primers used for PCR are shown in Table 1. PCR amplification system: 1 µL each of forward and reverse primers; 1 µL template; 25 µL Pfu enzyme (Tolu Harbor 2×MegaPfu Premix (with dye)); and ultrapure water to a final volume of 50 µL.

[0144] PCR reaction program: 95℃, 5 min; 95℃ for 30 s, TM temperature for 30 s, 72℃ for 30 s-1 min / kb, 32 cycles; 72℃, 5 min.

[0145] Table 2. Comparison of gene editing efficiency between mutant and wild-type effector proteins.

[0146]

[0147] The results showed that, at the OsPi21 target site in rice, the Casδ editing efficiency before modification was approximately 1% ( Figure 2 (Wt represents C in C), and the Casδ mutant Casδ-N915L of this invention exhibits enhanced editing activity at this target site, with an editing efficiency of approximately 30.6% (Wt represents C in C). Figure 2 N915L represents C. At the OsPDS target site, no editing activity was detected in the unmodified Casδ ( Figure 2 (WT represents B in Chinese), the editing efficiency of the modified Casδ-N915L is approximately 2% ( Figure 2 (N915L, represented by B). At the OsALS target, the unmodified Casδ editing efficiency was approximately 10% ( Figure 2 (WT represents A in Chinese), the editing efficiency of the modified Casδ-N915L is approximately 16% ( Figure 2 (Represented by N915L in A), the Casδ mutant Casδ-N915L of this invention also exhibits enhanced editing activity at this target.

[0148] In summary, the Casδ effector protein mutant Casδ-N915L obtained by screening in this invention has higher gene editing activity in rice compared with the wild-type Casδ effector protein, and can be applied to gene function research and breeding improvement in rice and other crops.

[0149] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A Cas effector protein mutant, characterized by: The Cas effector protein mutant is Casδ-N915L or a fusion protein containing Casδ-N915L, wherein Casδ-N915L is a protein whose amino acid sequence is sequence 3 in the sequence listing, the fusion protein is Casδ-N915L-NLS, and Casδ-N915L-NLS is a protein whose amino acid sequence is sequence 6 in the sequence listing.

2. A nucleic acid molecule, characterized by: The nucleic acid molecule encodes the Cas effector protein mutant of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is the encoding gene of the Cas effector protein mutant.

4. The nucleic acid molecule according to claim 3, characterized in that: The encoding gene is either the Casδ-N915L gene or the Casδ-N915L-NLS gene, the nucleotide sequence of the Casδ-N915L gene is sequence 2 in the sequence listing, and the nucleotide sequence of the Casδ-N915L-NLS gene is sequence 7 in the sequence listing.

5. An expression box, characterized in that: The expression cassette comprises the nucleic acid molecule as described in claim 2, 3 or 4.

6. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule as described in claim 2, 3 or 4.

7. Recombinant microorganisms, characterized by: The recombinant microorganism contains the Cas effector protein mutant of claim 1 or the nucleic acid molecule of claim 2, 3 or 4.

8. The application of proteins in rice nucleic acid editing, characterized by: The protein is the Cas effector protein mutant as described in claim 1.

9. A method for editing rice nucleic acids, characterized in that: The method includes contacting the rice genome with a guide RNA targeting the nucleic acid to be edited and the Cas effector protein mutant of claim 1 to achieve nucleic acid editing of the rice genome.

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