Application of chromosome regulating peptide structural domain in improving Cas protein editing efficiency

Abstract

The invention belongs to the field of nucleic acid editing, and particularly relates to the technical field of regular clustering interval short palindromic repeat (CRISPR). Specifically, the invention provides the application of the chromosome regulating peptide structural domain in improving the Cas protein editing efficiency, and the chromosome regulating peptide structural domain has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of gene editing, particularly to the field of regularly clustered short palindromic repeats (CRISPR) technology. Specifically, this invention relates to the application of chromosome regulatory peptide domains in improving the efficiency of Cas protein editing. Background Technology

[0002] CRISPR / Cas technology is a widely used gene editing technology that uses RNA to specifically bind to target sequences on the genome and cut DNA to create double-strand breaks, using biological non-homologous end joining or homologous recombination for site-specific gene editing.

[0003] This application utilizes the fusion of chromosome regulatory peptide domains with Cas enzymes, which improves the editing activity of Cas enzymes to a certain extent and has broad application prospects. Summary of the Invention

[0004] The inventors improved the editing activity and expanded the application range of Cas enzymes by fusing chromosome regulatory peptide domains into them.

[0005] On the one hand, the present invention provides the application of chromosome regulatory peptide domains in improving the editing efficiency of Cas proteins, or in the preparation of Cas proteins with improved editing efficiency.

[0006] In some embodiments, the chromosome regulatory peptide is derived from humans, and the chromosome regulatory peptide is an Alexandromys fortis high mobility group protein B1-like protein.

[0007] In some embodiments, the amino acid sequence of the chromosome regulatory peptide domain is shown in SEQ ID No. 2.

[0008] In some embodiments, the chromosome regulatory peptide is selected from any group I-III of the following:

[0009] I. The chromosome regulatory peptide is a human Alexandromys fortis high mobilitygroup protein B1-like protein. The amino acid sequence of the chromosome regulatory peptide domain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 2, and substantially retains the biological function of the chromosome regulatory peptide domain.

[0010] II. The chromosome regulatory peptide is a human Alexandromys fortis high mobilitygroup protein B1-like protein. Compared with SEQ ID No. 2, the amino acid sequence of the chromosome regulatory peptide domain has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids), and substantially retains the biological function of the chromosome regulatory peptide domain.

[0011] III. The chromosome regulatory peptide domain includes the amino acid sequence shown in SEQ ID No. 2.

[0012] On the other hand, the present invention provides a method for improving the efficiency of Cas protein editing, the method comprising the step of fusing the above-mentioned chromosome regulatory peptide domain with the Cas protein.

[0013] On the other hand, the present invention provides a fusion protein comprising a Cas protein and the aforementioned chromosome regulatory peptide domain.

[0014] In one embodiment, the Cas protein is a Cas12 family protein.

[0015] In one embodiment, the Cas protein is selected from one or any of Cas12i, Cas12j, Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, and Cas-sf0005.

[0016] In one embodiment, the Cas protein is a Cas protein of the Cas12i family, such as Cas12i1, Cas12i2, Cas12i3, or Cas12i12.

[0017] In a preferred embodiment, the Cas protein is Cas12i3, for example, the Cas12f.4 protein described in CN111757889B, or the Cas12i3 obtained by amino acid mutation described in Chinese patent applications with application numbers 2022103148077, 2022102697541, 2022106036073, 2022109432359, 2023100884374, 2023100667809, and 2023104503761.

[0018] In one embodiment, the Cas protein is the wild-type Cas12i3 or the mutant Cas12i3 described above.

[0019] In one embodiment, the Cas protein is a mutated Cas protein described in CN116004573B, preferably, the amino acid sequence of the Cas protein is shown in SEQ ID No. 5.

[0020] In one embodiment, the amino acid sequence of the Cas protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with SEQ ID No. 5.

[0021] In one embodiment, the amino acid sequence of the Cas protein, compared with SEQ ID No. 5, has one or more amino acid substitutions, deletions, or additions, for example, substitutions, deletions, or additions of 1-20 amino acids, or substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0022] In one embodiment, the Cas protein is a Cas protein of the Cas12j family, such as Cas12j19. Preferably, the Cas protein is the Cas12j.19 protein described in Chinese Patent CN111770992B, or the Cas12j19 obtained by amino acid mutation described in Chinese patent applications with application numbers 2023100922319 and 2023110952452.

[0023] In one embodiment, the Cas protein is Cas12a, for example, FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, or Lb4Cas12a; preferably, LbCas12a.

[0024] In some embodiments, the Cas protein is a natural wild-type Cas protein; in other embodiments, the Cas protein is an engineered Cas protein, for example, a Cas protein obtained through site-directed amino acid mutation.

[0025] In some embodiments, the chromosomal regulatory peptide domain is located at the N-terminus or C-terminus of the Cas protein; the regulatory peptide domain is linked to the N-terminus or C-terminus of the Cas protein via a linker.

[0026] The term "connector," as is well known in the art when referring to peptide linking, refers to a chemical group or molecule that links two molecules or parts. A connector may consist of a single linking molecule (e.g., a single amino acid) or may include more than one linking molecule. In some embodiments, the connector may be an organic molecule, group, polymer, or chemical part, such as a divalent organic part. In some embodiments, the connector may be an amino acid or a peptide.

[0027] The aforementioned linkers are well known in the art and include, but are not limited to, linkers containing one or more (e.g., 1, 2, 3, 4 or 5) amino acids (e.g., Glu or Ser) or amino acid derivatives (e.g., Ahx, β-Ala, GABA or Ava), or PEG, etc.

[0028] In some embodiments, the linker may be a GS linker. In some embodiments, the linker may comprise the amino acid sequence (GGS)n, GS, SG, GSSG, S(GGS)n, SGGS, or (GGGGS)n, where n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker may comprise the amino acid sequence: SGGSGGSGGS. In some embodiments, the linker may comprise the amino acid sequence: SGSETPGTSESATPES, also known as an XTEN linker. In some embodiments, the linker may comprise the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGS, also known as a GS-XTEN-GS linker. Preferably, the amino acid sequence of the linker is as shown in SEQ ID No. 6.

[0029] In this invention, the amino acid site refers to the site starting from the N-terminus of the amino acid sequence.

[0030] The present invention also provides a fusion protein, which includes the fusion protein as described above and other modified portions.

[0031] In one embodiment, the modified portion is selected from other proteins or peptides, detectable markers, or any combination thereof.

[0032] In one embodiment, the modified portion is selected from epitope tags, reporter gene sequences, nuclear localization signal (NLS) sequences, targeting portions, transcriptional activation domains (e.g., VP64), transcriptional repression domains (e.g., KRAB or SID domains), nuclease domains (e.g., Fok1), and domains having activities selected from: nucleotide deaminases (e.g., adenosine deaminase or cytidine deaminase), methyltransferase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional releasing factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity; and any combination thereof. The NLS sequences are well known to those skilled in the art, and examples include, but are not limited to, the SV40 large T antigen, EGL-13, c-Myc, and TUS protein.

[0033] In one embodiment, the NLS sequence is located at, near, or close to the end (e.g., N-terminus, C-terminus, or both ends) of the Cas protein of the present invention.

[0034] The epitope tag is well known to those skilled in the art, including but not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art can choose other suitable epitope tags (e.g., for purification, detection or tracing).

[0035] The reporter gene sequences are well known to those skilled in the art, and examples include, but are not limited to, GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.

[0036] In one embodiment, the fusion protein of the present invention includes a domain capable of binding to DNA molecules or intracellular molecules, such as maltose-binding protein (MBP), the DNA-binding domain (DBD) of Lex A, the DBD of GAL4, etc.

[0037] In one embodiment, the fusion protein of the present invention contains a detectable marker, such as a fluorescent dye, such as FITC or DAPI.

[0038] In one embodiment, the fusion protein of the present invention is optionally coupled, conjugated, or fused to the modified portion via a linker.

[0039] In one embodiment, the modified portion is directly connected to the N-terminus or C-terminus of the fusion protein or Cas protein of the present invention.

[0040] In one embodiment, the modified portion is attached to the N-terminus or C-terminus of the fusion protein or Cas protein of the present invention via a linker. Such linkers are well known in the art, and examples include, but are not limited to, linkers containing one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., Glu or Ser) or amino acid derivatives (e.g., Ahx, β-Ala, GABA, or Ava), or PEG, etc.

[0041] The fusion protein of the present invention is not limited by its production method; for example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0042] On the other hand, the present invention provides an isolated polynucleotide comprising:

[0043] (a) The polynucleotide sequence encoding the fusion protein of the present invention;

[0044] Alternatively, a polynucleotide complementary to the polynucleotide described in (a).

[0045] In one embodiment, the nucleotide sequence is codon-optimized for expression in prokaryotic cells. In another embodiment, the nucleotide sequence is codon-optimized for expression in eukaryotic cells.

[0046] In one embodiment, the cell is an animal cell, such as a mammalian cell.

[0047] In one embodiment, the cell is a human cell.

[0048] In one embodiment, the cell is a plant cell, such as the cell of a cultivated plant (e.g., cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.

[0049] In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

[0050] On the other hand, the present invention provides a gRNA comprising a first segment and a second segment; the first segment is also referred to as a "backbone region", "protein binding region", "protein binding sequence", or "direct repeat sequence"; the second segment is also referred to as a "target sequence for targeting nucleic acids", "target segment for targeting nucleic acids", or "guide sequence for targeting target sequences".

[0051] The first segment of the gRNA can interact with the Cas protein of the present invention, thereby enabling the Cas protein and gRNA to form a complex.

[0052] In a preferred embodiment, the first segment is a repeating sequence in the same direction as described above.

[0053] The target sequence or target region of the nucleic acid targeted by this invention comprises a nucleotide sequence complementary to a sequence in the target nucleic acid. In other words, the target sequence or target region of the nucleic acid targeted by this invention interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the target sequence or target region of the nucleic acid can be altered or modified to hybridize with any desired sequence within the target nucleic acid. The nucleic acid is selected from DNA or RNA.

[0054] The percentage of complementarity between the target sequence or target region of the target nucleic acid and the target sequence of the target nucleic acid may be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).

[0055] The "backbone region," "protein-binding region," "protein-binding sequence," or "direct repeat sequence" of the gRNA of this invention can interact with CRISPR proteins (or Cas proteins). The gRNA of this invention guides the interacting Cas protein to a specific nucleotide sequence within the target nucleic acid through the targeting sequence of the target nucleic acid.

[0056] Preferably, the guide RNA comprises a first segment and a second segment in the 5' to 3' direction.

[0057] In this invention, the second segment can also be understood as a guide sequence for hybridization with the target sequence.

[0058] The gRNA of the present invention can form a complex with the Cas protein.

[0059] The present invention also provides a carrier comprising, as described above, a fusion protein, an isolated nucleic acid molecule or a polynucleotide; preferably, it further comprises a regulatory element operatively linked thereto.

[0060] In one embodiment, the regulatory element is selected from one or more of the following: enhancers, transposons, promoters, terminators, leader sequences, polyadenylation sequences, and marker genes.

[0061] In one embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector.

[0062] In some implementations, the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated vectors, and herpes simplex vectors), and may also be plasmids, viruses, granules, bacteriophages, etc., which are well known to those skilled in the art.

[0063] On the other hand, the present invention provides a composition comprising:

[0064] (i) Protein components selected from: the engineered fusion protein described above; and

[0065] (ii) A nucleic acid component comprising (a) a guide sequence capable of hybridizing with a target sequence; and (b) a unidirectional repeat sequence capable of binding to the Cas protein in the fusion protein of the present invention.

[0066] The protein components and nucleic acid components combine to form a complex.

[0067] In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.

[0068] In one embodiment, the complex or composition is non-natural or modified. In one embodiment, at least one component of the complex or composition is non-natural or modified. In one embodiment, the first component is non-natural or modified; and / or, the second component is non-natural or modified.

[0069] On the other hand, the present invention provides an engineered host cell comprising the above-described fusion protein, or the above-described polynucleotide, or the above-described vector, or the above-described composition.

[0070] In some implementations, the cell is a prokaryotic cell.

[0071] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a non-human mammalian cell, such as cells of non-human primates, cattle, sheep, pigs, dogs, monkeys, rabbits, or rodents (such as rats or mice). In some embodiments, the cell is a non-mammalian eukaryotic cell, such as cells of poultry (such as chickens), fish, or crustaceans (such as clams or shrimp). In some embodiments, the cell is a plant cell, such as cells of monocotyledonous or dicotyledonous plants, or cells of cultivated plants or food crops such as cassava, corn, sorghum, soybeans, wheat, oats, or rice, such as algae, trees, or productive plants, fruits, or vegetables (e.g., trees such as citrus trees, nut trees; nightshade plants, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).

[0072] In some implementations, the cell is a stem cell or stem cell line.

[0073] In some cases, the host cells of the present invention contain genetic or genomic modifications that are not present in their wild type.

[0074] The present invention also provides the use of the above-mentioned fusion protein, or the above-mentioned polynucleotide, or the above-mentioned vector, or the above-mentioned composition, or the above-mentioned host cell in gene editing; or, in the preparation of reagents or kits for gene editing.

[0075] The present invention also provides a method for editing a target nucleic acid, the method comprising contacting the target nucleic acid with the aforementioned fusion protein, or the aforementioned polynucleotide, or the aforementioned vector, or the aforementioned composition, or the aforementioned host cell.

[0076] In one embodiment, the method is to edit, target, or cleave target nucleic acids intracellularly or extracellularly.

[0077] The gene editing includes modifying genes, knocking out genes, altering the expression of gene products, repairing mutations, and / or inserting polynucleotides, and gene mutations.

[0078] The editing can be performed in prokaryotic and / or eukaryotic cells.

[0079] On the other hand, the present invention also provides a kit for gene editing, the kit comprising the above-mentioned fusion protein, or the above-mentioned polynucleotide, or the above-mentioned vector, or the above-mentioned composition, or the above-mentioned host cell.

[0080] Beneficial effects of the invention

[0081] This invention fuses the chromosome regulatory peptide domain with the Cas protein, thereby enhancing the activity of the Cas protein and showing broad application prospects. Attached Figure Description

[0082] Figure 1 This study investigates the effect of fusing different chromosome regulatory peptides on the editing efficiency of Cas-SF01.

[0083] Figure 2 These are the results of the Cas-SF01 fusion with HB1 regulatory peptide editing efficiency test.

[0084] Figure 3 These are the results of the editing efficiency test of Cas-SF01 fused with HB1 regulatory peptide in soybean.

[0085] The sequence information involved in this invention is as follows:

[0086] Detailed Implementation

[0087] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0088] Example 1. Obtaining Chromosomal Regulatory Peptides

[0089] Bioinformatics analysis was used to obtain different types of chromosome regulatory peptides. In this embodiment, the chromosome regulatory peptide domains HN1, HB1, H1G, and CHD1 were selected, and their amino acid sequences are shown in SEQ ID No. 1-4, respectively.

[0090]

[0091] Example 2. Editing effect of different chromosome regulatory peptide domains with Cas-SF01 fusion protein in animal cells Rate

[0092] In this embodiment, Cas-SF01 (a Cas protein BC26312 with amino acid mutations is disclosed in CN116004573B, which is referred to as Cas-SF01 in this embodiment, and the amino acid sequence of Cas-SF01 is shown in SEQ ID No. 5) was fused with the chromosome regulatory peptide domain in Example 1, and its editing activity was detected.

[0093] Specifically, the chromosome regulatory peptide domains HN1, HB1, H1G, and CHD1 from Example 1 were respectively used with XTENlinker (amino acid sequence as shown in SEQ ID No. 6; other linkers in the art, such as GGGS, can also be used). n(e.g.,) are linked to the N-terminus of Cas-SF01 to form fusion proteins HN1-Cas-SF01, HB1-Cas-SF01, H1G-Cas-SF01, and CHD1-Cas-SF01. The editing efficiency was tested using Cas-SF01 protein as a control. Target sites were designed for the TTR gene in 239T cells, and 12 target sites (TTR-13, TTR-23, TTR-1, TTR-2, TTR-3, TTR-4, TTR-11, TTR-14, TTR-16, TTR-19, TTR-25, and TTR-27) were selected for editing efficiency testing. The vector pcDNA3.3 was modified to carry EGFP fluorescent protein and the PuroR resistance gene. The SV40 NLS-Cas fusion protein was inserted via the XbaI and PstI restriction sites; the U6 promoter and gRNA sequence were inserted via the Mfe1 restriction site. The CMV promoter initiates the expression of the fusion protein SV40 NLS-Cas-XX-NLS-GFP. The proteins Cas-XX-NLS and GFP are linked using the linker peptide T2A. The EF-1α promoter initiates the expression of the puromycin resistance gene. Plating: CHO cells are plated to a confluence of 70-80% at a density of 8*10^4 cells / well in 12-well plates. Transfection: Transfection is performed 24 hours after plating. 6.25 µl of Hieff Trans™ liposome nucleic acid transfection reagent is added to 100 µl opti-MEM and mixed well; 2.5 µg of plasmid is added to 100 µl opti-MEM and mixed well. The diluted Hieff Trans™ liposome nucleic acid transfection reagent and diluted plasmid are mixed thoroughly and incubated at room temperature for 20 min. The incubated mixture is then added to cell-coated culture medium for transfection. Puromycin selection: Puromycin is added 24 hours after transfection to a final concentration of 10 μg / ml. After 24 hours of treatment with puromycin, the culture medium was replaced with normal medium and cultured for another 24 hours. 48 hours after transfection, cells were digested with trypsin-EDTA (0.05%) and sorted using flow cytometry (FACS) for cells exhibiting GFP signals.

[0094] DNA extraction, PCR amplification of the area near the editing region, and hiTOM sequencing: Cells were collected after trypsin digestion, and genomic DNA was extracted using a cell / tissue genomic DNA extraction kit (Biotech). Genomic DNA was amplified in the region near the target site. PCR products were then sequenced using hiTOM. Sequencing data were analyzed, and the types and proportions of sequences within a 15nt upstream and 10nt downstream of the target site were statistically analyzed. Sequences with a SNV frequency greater than or equal to 1% or a non-SNV mutation frequency greater than or equal to 0.06% were statistically analyzed to obtain the editing efficiency of different fusion proteins (HN1-Cas-SF01, HB1-Cas-SF01, H1G-Cas-SF01, and CHD1-Cas-SF01) on the target sites (TTR-13, TTR-23, and TTR-1). Results are as follows: Figure 1 As shown: Figure 1 Different dots represent editing efficiency at a specific target site, where SF01 is Cas-SF01 without fusion with the chromosome regulatory peptide; HN1-SF01, HB1-SF01, H1G-SF01, and CHD1-SF01 are the fusion proteins HN1-Cas-SF01, HB1-Cas-SF01, H1G-Cas-SF01, and CHD1-Cas-SF01, respectively. The results show that compared to Cas-SF01, the editing efficiency of the fusion protein HB1-Cas-SF01 is significantly improved, while the editing efficiency of the fusion proteins HN1-Cas-SF01, H1G-Cas-SF01, and CHD1-Cas-SF01 shows no significant change.

[0095] The fusion protein HB1-Cas-SF01 ( Figure 2 HB1-SF01 and Cas-SF01 (in the middle) Figure 2 The editing efficiency of SF01 in the above 12 targets was compared, and the results are as follows: Figure 2 As shown: Figure 2 Different points represent editing efficiency at a specific target site. The average editing efficiency of the fusion protein HB1-Cas-SF01 is about 20% higher than that of Cas-SF01.

[0096] The target sequence and primer information for the TTR gene gRNA are as follows:

[0097]

[0098] Example 3. Editing efficiency of CasSF01 fusion protein with different chromosome regulatory peptide domains in plants

[0099] The editing efficiency of the fusion protein HB1-Cas-SF01 in Example 2 in soybeans was tested, using Cas-SF01 protein as a control.

[0100] Gene editing in soybeans can be performed using the aforementioned fusion proteins HB1-Cas-SF01 and Cas-SF01 in a manner known in the art. In this embodiment, the method used is as follows:

[0101] 1. Construction of gene editing vectors

[0102] Based on the coding sequence of the soybean gene, gRNAs targeting the Cas protein were designed. The designed gRNA target sequences (guide sequences) are shown in the table below.

[0103] Four soybean genes (Lox2, FAD2-1a, FAD2-1b and miR396a) were selected as target sites for gRNA design. The designed gRNA target sequences (guide sequences) and primers are shown in the table below.

[0104]

[0105] Based on the homologous repeat sequence of Cas-SF01 gRNA, a gRNA containing both a homologous repeat sequence and a guide sequence was designed. Annealing primers were designed according to the target site. After primer annealing, the gene editing backbone vector was ligated using the Golden Gate method to obtain the gene editing vector.

[0106] 2. Obtaining recombinant bacteria

[0107] 1) Transformation of Escherichia coli

[0108] The gene-editing vector from step 1 is transformed into E. coli. The transformed E. coli are subjected to bacterial culture PCR. The amplified products with the correct PCR band size are sequenced. The E. coli with the correct sequencing results are recombinant E. coli containing the gene-editing vector.

[0109] 2) Transformation of Agrobacterium

[0110] After culturing the recombinant E. coli containing the gene editing vector in step 1), plasmid DNA was extracted, added to Agrobacterium competent cells, and placed in an ice bath for 5 min, liquid nitrogen for 5 min, a 37°C water bath for 5 min, and then placed on ice for 5 min.

[0111] Remove the centrifuge tube, add 700µl of culture medium (antibiotic-free), and incubate at 28°C with shaking for 2-4 hours;

[0112] Take out the bacterial culture and spread it onto a medium plate containing the corresponding antibiotic. Incubate it upside down in an incubator. Colonies will be visible after about 2 days. Perform PCR on the colonies according to the method in step 1), and sequence the amplified products. Agrobacterium with correct sequencing results is recombinant Agrobacterium containing the gene editing vector.

[0113] 3. Soybean genetic transformation

[0114] Soybean genetic transformation was performed using conventional methods in the field. Soybeans were transformed using gene-editing vectors containing the fusion protein HB1-Cas-SF01 or Cas-SF01 and the aforementioned gRNA. Sterilized soybean seeds were sown in a sterile, moist substrate of 2:1 mixture of nutrient soil and vermiculite at a depth of 1 cm. Plants were cultured in a greenhouse under conditions of 25°C, 70% relative humidity, and a 12-hour light / 12-hour dark cycle for 7-14 days. A 1 cm oblique incision was made at the hypocotyl of the soybean seedling using a sterile scalpel. Agrobacterium rhizogenes K599 bacterial suspension containing the expression vector was directly applied to the oblique incision surface. The seedlings were then planted in sterile vermiculite pots and irrigated with 10 ml of Agrobacterium rhizogenes K599 bacterial suspension. A high-humidity environment was maintained for 16 days to promote the growth of hairy roots. Finally, genomic DNA was extracted from the hairy roots and analyzed for editing efficiency using PCR amplification.

[0115] 4. Results

[0116] Soybeans were transformed using gene editing vectors containing the fusion protein HB1-Cas-SF01 or Cas-SF01 and the aforementioned gRNA. Positive seedlings were screened, and the target gene of the positive seedlings was subjected to Sanger sequencing. Editing efficiency was statistically analyzed. Results are as follows: Figure 3 As shown, with Cas-SF01 ( Figure 3 Compared to SF01 in the original text, the fusion protein HB1-Cas-SF01 ( Figure 3 The editing efficiency of HB1-SF01 at the four sites in soybean was significantly improved, with an improvement of 15-75%.

[0117] The above results indicate that fusing the chromatin regulatory peptide domain with the Cas protein can significantly improve the editing efficiency of the Cas protein.

Claims

1. The application of chromosome regulatory peptide domains in improving the efficiency of Cas protein editing, or in the preparation of Cas proteins with improved editing efficiency; characterized in that, The chromosome regulatory peptide is selected from any one of the following groups I-III: I. The chromosome regulatory peptide is a human Alexandromys fortis high mobility groupprotein B1-like protein. The amino acid sequence of the chromosome regulatory peptide domain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 2, and substantially retains the biological function of the chromosome regulatory peptide domain. II. The chromosome regulatory peptide is a human Alexandromys fortis high mobility groupprotein B1-like protein. Compared with SEQ ID No. 2, the amino acid sequence of the chromosome regulatory peptide domain has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or additions), and the biological function of the chromosome regulatory peptide domain is substantially preserved. III. The chromosome regulatory peptide domain includes the amino acid sequence shown in SEQ ID No.

2.

2. A method for improving the efficiency of Cas protein editing, the method comprising the step of fusing the chromosome regulatory peptide domain of claim 1 with the Cas protein.

3. A fusion protein comprising a Cas protein and the chromosome regulatory peptide domain of claim 1, wherein the Cas protein is a Cas12 family protein; Preferably, the chromosome regulatory peptide domain is located at the N-terminus of the Cas protein.

4. An isolated polynucleotide, characterized in that, The polynucleotide is a polynucleotide sequence encoding the fusion protein of claim 3.

5. A carrier, characterized in that, The vector comprises the polynucleotide of claim 4 and a regulatory element operatively linked thereto.

6. A composition, characterized in that, The composition comprises: (i) a protein component selected from the fusion protein of claim 3; (ii) A nucleic acid component, which is gRNA, said gRNA being capable of binding the Cas protein in the fusion protein of claim 3; The protein components and nucleic acid components combine to form a complex.

7. An engineered host cell, characterized in that, The host cell comprises the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the composition of claim 6.

8. The use of the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the composition of claim 6, or the host cell of claim 7 in gene editing; or, in the preparation of a kit for gene editing.

9. A method for editing a target nucleic acid, the method comprising contacting the target nucleic acid with a fusion protein of claim 3, or a polynucleotide of claim 4, or a vector of claim 5, or a composition of claim 6, or a host cell of claim 7.

10. A kit for gene editing, the kit comprising the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the composition of claim 6, or the host cell of claim 7.

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