Effect nuclease and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to novel effect nuclease and application thereof, according to found and researched Cas12a effect nuclease sequences, the novel effect nuclease related to two regular clustered interval short palindromic repeats (CRISPR) is obtained through design, screening and functional verification, the invention particularly provides a Cas protein, the Cas protein is Cas-kf8 or Cas-kf9, and the Cas-kf8 or the Cas-kf9 is a Cas-kf8 protein or a Cas-kf9 protein or a Cas-kf9 protein or a Cas-kf9 protein or a Cas-kf9 protein or a Cas-kf9 protein or a Cas-kf9 protein. The amino acid sequence of the Cas-kf8 is as shown in SEQ ID No.1, the amino acid sequence of the Cas-kf9 is as shown in SEQ ID No.4, and the Cas protein provided by the invention shows the activity of nuclease through experiments and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to novel effector nucleases Cas-kf8 and Cas-kf9 associated with clustered regularly interspaced short palindromic repeats (CRISPR), and their application in the field of gene editing. Background Art

[0002] Gene editing technology has developed rapidly in recent years. It uses the effector nuclease Cas (CRISPR-associated) associated with the clustered regularly interspersed short palindromic repeats (CRISPR) in the bacterial acquired immune system to cut specific genes in cells at pre-selected sites in the genome. Through two DNA repair mechanisms inherent in cells, non-homologous end joining (NHEJ) and homology dependent repair (HDR), the breaks are repaired to ensure cell survival. NHEJ has high repair efficiency and does not require a template. The restoration of the linking process at the cut point will introduce insertion or deletion mutations, thereby inactivating the gene, which is dominant; HDR requires a repair template and achieves precise repair according to the template, which accounts for a very small proportion. If a pre-designed DNA fragment is provided as a template, the fragment sequence can be inserted into the pre-selected site to modify the target gene, but the efficiency is extremely low, which seriously restricts its widespread application.

[0003] The gene editing technology based on the CRISPR effector nuclease SpCas9 of Streptococcus pyogenes clustered short palindromic repeats was first used to achieve in vivo gene editing in 2013 (Cong et al., 2013, Science 339, 819-823; Mali et al., 2013, Science 339, 823-826). The Cas9 endonuclease forms a nucleoproteinase complex with the guide gRNA, which recognizes the target by base pairing between the 20 bp-long RNA sequence in the gRNA and the edited DNA sequence. It has high specificity, strong enzyme cleavage activity, and simple experimental design, making the CRISPR / Cas9 gene editing system quickly and successfully applied in different animal and plant systems. It is also the preferred method for plant breeding using gene editing technology, and has achieved knockout editing of various plants mainly through the NHEJ pathway and a few HDR-mediated precision design editing (Li et al., 2015, Plant Physiol 169, 960-970; Ma et al., 2015, Plant Physiol 169, 960-970). al., 2016, Mol Plant 9, 961-974; Chen et al., 2019, Annu Rev Plant Biol 70, 667-97; Mao et al., 2019, Natl Sci Rev 6, 421-437). Combined with multiple gRNA synchronous expression technology, CRISPR / Cas9 technology can simultaneously perform efficient NHEJ pathway editing on multiple genes and multiple sites, achieving synchronous improvement of multiple traits (Xie et al., 2015, Proc Natl Acad Sci USA 112, 3570-3575; Hsieh-Feng and Yang 2020, aBIOTECH, doi.org / 10.1007 / s42994-019-00014-w). By fusing Cas9 with other proteases such as deaminases or reverse transcriptases, precise editing of single bases or small fragments of DNA sequences can be achieved (Kim et al., 2017, Nat Biotechnol 35, 371-376; Komor et al., 2016, Nature 533, 420-424; Nishida et al., 2016, Science 353, aaf8729; Gaudelli et al., 2017, Nature 551, 464-471; Anzalone et al., 2019, Nature 576, 149-157).By mutating the SpCas9 protein sequence, mutants that recognize PAM (Protospacer Adjacent Motif) sequences other than NGG can be obtained, expanding the gene sites that can be edited (Hu et al., 2018, Nature 556, 57-63; Kleinstiver et al., 2015, Nature 523, 481-485; Nishimasu et al., 2018, Science 361, 1259-1262; Walton et al., 2020, Science 368, 290-296). By fusing Cas9 with other effector protein functional groups such as transcription factors, regulation of gene expression levels can be achieved (Pan et al., 2021, Curr Opinion Plant Biol 60, 101980).

[0004] Acquired immune systems containing CRISPR structures are widely present in bacteria and archaea. According to the number of proteins involved in immune interference reactions, they are divided into two categories: 1 and 2. The 1st category system requires multiple proteins to form a complex to perform immune interference functions, while the 2nd category can achieve interference with only one protease containing a multifunctional group (Makarova et al., 2020, Nat Rev Microbiol 18, 67-83; Nidhi et al., 2021, Int J Mol Sci 22, 3327). Each type of system is divided into multiple types according to structure, composition, etc. The commonly used SpCas9 is a typical representative of the 2nd category II type CRISPR system, which requires the 3' end of the target sequence to have a conservative NGG as the PAM sequence and performs blunt end cutting on the target sequence (Jinek et al., 2012, Science 337, 816-821). Effector nucleases of other two types of type II CRISPR systems, such as SaCas9 from Staphylococcus aureus and NmeCas9 from Neisseria meningitidis, have also been successfully used for editing (Ran et al., 2015, Nature 520, 186-191; Amrani et al., 2018, Genome Biol 19, 214).

[0005] The Cas12 effector nucleases of the V-type CRISPR system belonging to the same category 2, such as FnCpfI and LbCpf1 (hereinafter referred to as FnCas12a and LbCas12a) derived from the bacterial strain Francisella tularensis subsp.Novicida U112 and Lachnospiraceae bacterium ND2006, require the 5' end of the target sequence to have a conservative TTTN as a PAM sequence and perform sticky end cutting on the target sequence (Zetsche et al., 2015, Cell 163, 1-13). The Cas12a nuclease of other 2 types of V-type CRISPR systems has also been used for gene editing to varying degrees, most of which mainly recognize target DNA sequences containing a conservative PAM sequence of TTTV (V is A, C, or G) at the 5' end, and cut the DNA double strand at the distal end of the PAM to produce sticky ends. In addition, the Cas12a effector nuclease also has RNase function, which can autonomously process and cut the expression precursor pre-crRNA of the CRISPR repeat array to form a single mature crRNA, guiding the recognition of the target DNA sequence.

[0006] Although Cas12a effector nucleases have been identified and discovered in many bacteria based on the principle of homology, the homology between protease sequences from different sources is not high, and enzymes with distant genetic distances often have only about 30% amino acid sequence similarity. In addition to the difficulty in directly judging the biological function of a specific Cas12a enzyme based on the sequence due to low homology, not every enzyme is an active protein with immune function. Some Cas12a immune systems are incomplete in themselves, lacking other necessary components such as Cas1, Cas2, Cas4 and other auxiliary proteins, or CRISPR arrays are missing (Yan et al., 2019, Science 363, 88-91). Even if the Cas12a enzymes with complete composition and sequence do not necessarily have enzymatic activity or even gene editing functions of eukaryotic organisms, only a few may remain active after expression in eukaryotic organisms, so they must be verified through specific experimental screening. Zetsche et al. conducted detailed experimental studies on a total of 16 Cas12a effector nucleases, including FnCpfI and LbCpf1, of which only 8 showed effective activity in in vitro DNA cleavage experiments. The researchers then optimized the codons of these 8 effector nucleases and expressed them in human cell lines. Only 2 were detected to have effective activity and achieved editing of the target gene (Zetsche et al., 2015, Cell 163, 1-13).

[0007] The CRISPR / Cas gene editing systems discovered so far have different characteristics. Based on a variety of different reasons and complex and diverse research and application needs, the continuous development of new gene editing tool enzymes is of great significance to the development and application research of biotechnology. Summary of the invention

[0008] According to the discovered and studied Cas12a effector nuclease sequence, the present invention designs, screens and performs functional verification to obtain two new effector nucleases Cas-kf8 and Cas-kf9. Based on this discovery, the present invention develops a new CRISPR / Cas system and a gene editing method based on the system.

[0009] Cas proteins

[0010] On the one hand, the present invention provides a Cas protein (or, referred to as "effector nuclease", "Cas enzyme" or "effector protein"), which is an effector protein in the CRISPR / Cas system, and the Cas protein is Cas-kf8 or Cas-kf9, and the Cas-kf8 amino acid sequence is shown in SEQ ID No 1, and the Cas-kf9 amino acid sequence is shown in SEQ ID No 4.

[0011] Compared with the sequence of SEQ ID No 1 or 4, in some embodiments, the Cas protein is a sequence having one or more amino acid substitutions, deletions or additions, and substantially retains its biological function derived from the sequence; the one or more amino acids include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or additions; in some embodiments, the amino acid sequence has at least 50% sequence identity with the sequence shown in SEQ ID No 1 or 4, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity, and substantially retains its biological function derived from the sequence.

[0012] It is clear to those skilled in the art that the primary structure of a protein can be changed without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions are clear to those skilled in the art. Specifically, the amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, i.e., another non-polar amino acid residue can be replaced with a non-polar amino acid residue, another polar non-charged amino acid residue can be replaced with a polar uncharged amino acid residue, another basic amino acid residue can be replaced with a basic amino acid residue, and another acidic amino acid residue can be replaced with an acidic amino acid residue. The amino acid residues replaced in this way may or may not be encoded by the genetic code. As long as the substitution does not result in the inactivation of the biological activity of the protein, the conservative substitution of one amino acid replaced by other amino acids belonging to the same group falls within the scope of the present invention. In addition, the present invention also covers proteins comprising one or more other non-conservative amino acid substitutions, as long as the non-conservative substitution does not significantly affect the desired function and biological activity of the protein of the present invention.

[0013] The functions and activities include, but are not limited to, activity binding to gRNA, endonuclease activity, and activity binding to and cutting a specific site of the target sequence under the guidance of gRNA, including but not limited to Cis cutting activity and Trans cutting activity.

[0014] As is well known in the art, one or more amino acid residues can be changed (replaced, deleted, truncated or inserted) from the amino N and / or carboxyl C-terminus of a protein while still retaining its functional activity. Therefore, proteins in which one or more amino acid residues are changed from the N and / or C-terminus of the Cas protein of the present invention while retaining its desired functional activity are also within the scope of the present invention. These changes may include changes introduced by modern molecular methods such as PCR, which includes PCR amplification of a protein coding sequence by means of including an amino acid coding sequence in an oligonucleotide used in PCR amplification to change or extend the protein coding sequence.

[0015] It should be recognized that proteins can also be changed in various ways, including amino acid substitutions, deletions, truncations and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of Cas proteins can be prepared by mutations to DNA. It can also be accomplished by other forms of mutagenesis and / or by directed evolution, for example, using known mutagenesis, recombination and / or shuffling methods, combined with related screening methods, to perform single or multiple amino acid substitutions, deletions and / or insertions.

[0016] Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the present invention can occur (e.g., naturally occurring mutations) or be produced (e.g., using rDNA technology, recombinant DNA) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domain of the protein, the properties of the polypeptide may be changed, but the polypeptide may retain its activity. If the mutations present are not close to the catalytic domain, active site, or other functional domain, a smaller effect can be expected.

[0017] The present invention also provides a fusion protein, which includes a Cas protein having an amino acid sequence of SEQ ID No 1 (Cas-kf8) or SEQ ID No 4 (Cas-kf9) and other modified parts.

[0018] In one embodiment, the modifying moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.

[0019] In one embodiment, the modifying portion is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting portion, a transcriptional activation domain (e.g., VP64), a transcriptional repression domain (e.g., a KRAB domain or a SID domain), a nuclease domain (e.g., Fok1), and a domain having an activity selected from the following: nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release 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.

[0020] The NLS (Nuclear Localization Signal) nuclear localization sequence is well known to those skilled in the art, and examples thereof include but are not limited to SV40 large T antigen, Nucleoplasmin, EGL-13, c-Myc and TUS protein.

[0021] In one embodiment, the NLS sequence is located at, near or close to a terminus (e.g., the N-terminus, the C-terminus, or both) of the Cas protein of the invention.

[0022] 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 select other suitable epitope tags (e.g., purification, detection or tracing).

[0023] The reporter gene sequence is well known to those skilled in the art, and examples thereof include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.

[0024] In one embodiment, the fusion protein of the invention comprises a detectable label, such as a fluorescent dye, such as FITC or DAPI.

[0025] In one embodiment, the Cas protein of the present invention is optionally coupled, conjugated or fused to the modified portion via a linker.

[0026] In one embodiment, the modified portion is connected to the N-terminus or C-terminus of the Cas protein of the present invention via a linker. Such linkers are well known in the art, and examples thereof include but are not limited to linkers comprising 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.

[0027] The Cas protein, protein derivative or fusion protein of the present invention is not limited by the way it is produced. For example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0028] Cas protein nucleic acid

[0029] On the other hand, the present invention provides an isolated polynucleotide comprising a polynucleotide sequence encoding the Cas protein or fusion protein of the present invention.

[0030] In one embodiment, the polynucleotide sequence is codon optimized for expression in prokaryotes. In one embodiment, the polynucleotide sequence is codon optimized for expression in eukaryotic cells.

[0031] In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

[0032] In a specific embodiment, the polynucleotide sequence provided by the present invention is shown as SEQ ID No 2 or 5.

[0033] In some embodiments, the polynucleotide sequence has a sequence with one or more base substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more base substitutions, deletions or additions) compared to the sequence shown in SEQ ID No 2 or 5.

[0034] Those skilled in the art are aware that, based on the degeneracy in the genetic code, most amino acids are encoded by multiple different codons. The genetic code is the process in which the genetic information on DNA or mRNA is translated into proteins in an organism, wherein each amino acid is encoded by one or more specific three nucleic acid sequences (codons). Based on this property, different nucleic acid sequences (codons) can be transcribed and translated into the same amino acid. As long as changes in the polynucleotide sequence do not result in translation into different amino acids or inactivation of the biological activity of the protein, the polynucleotide sequence falls within the scope of the present invention.

[0035] Carrier

[0036] The present invention also provides a vector, which comprises the Cas protein and the isolated polynucleotide as described above; preferably, it also includes a regulatory element operably linked thereto.

[0037] In one embodiment, the regulatory element is selected from one or more of the following groups: enhancer, transposon, promoter, terminator, leader sequence, polyadenylation sequence, marker gene.

[0038] In one embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector, which are known to those skilled in the art and may be naturally occurring or artificially synthesized vector structures. In a specific embodiment, the present invention provides commercially available vectors pACYC-Duet-1 and pUC19.

[0039] Vector system

[0040] The present invention provides an engineered non-naturally occurring vector system, or a CRISPR-Cas system, which includes a Cas protein or a nucleic acid sequence encoding the Cas protein and a nucleic acid encoding one or more gRNAs, wherein the gRNA includes a repeat sequence that binds to the Cas protein and a spacer sequence for targeting a target sequence, and the spacer sequence is complementary to the target sequence.

[0041] In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more gRNAs are artificially synthesized.

[0042] In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more gRNAs do not co-exist naturally.

[0043] The one or more gRNAs target one or more target sequences in the cell. The one or more gRNAs hybridize with the target sequence of the genomic locus of the DNA molecule encoding one or more gene products, guide the Cas protein to the genomic locus of the DNA molecule of the one or more gene products, and the Cas protein modifies, edits or cuts the target sequence after reaching the target sequence position, thereby changing or modifying the expression of the one or more gene products.

[0044] The cells of the present invention include one or more of animals, plants or microorganisms.

[0045] In some embodiments, the Cas protein is codon optimized for expression in a cell.

[0046] In some embodiments, the Cas protein directs cleavage of one or both nucleic acid strands at the location of the target sequence.

[0047] In a specific embodiment, the repetitive sequence designed by the present invention to bind to the Cas protein is shown as SEQ ID No 3 or 6.

[0048] The present invention also provides an engineered non-naturally occurring vector system, which may include one or more vectors, which include: a gRNA expression unit and the Cas protein expression unit, and the gRNA expression unit and the Cas protein expression unit are located on the same or different vectors of the system.

[0049] The expression unit is linked to various regulatory elements including a promoter (e.g., a constitutive promoter or an inducible promoter), an enhancer (e.g., a 35S promoter or a 35S enhanced promoter), an internal ribosome entry site (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation PolyA signals and poly PolyU sequences).

[0050] In one embodiment, the gRNA expression unit is ZmU6 pro (promoter): gRNA: AtU6-26 term. Furthermore, a BsaI restriction site is left between ZmU6 pro and gRNA, and one or more gRNA sequences can be inserted to form a gene editing vector system targeting any target site of the target gene.

[0051] In one embodiment, the Cas protein expression unit is ZmUBI1 pro (promoter): Cas (Cas-kf8 or Cas-kf9): PsE9 term (terminator).

[0052] In one embodiment, the vector system further comprises a resistance screening gene expression unit, wherein the resistance screening gene is used in the genetic transformation process to screen out successfully transformed cells or organisms, examples of which include but are not limited to marker genes such as hygromycin resistance gene (HYG), neomycin resistance gene, tetracycline resistance gene, penicillin resistance gene, streptomycin resistance gene, herbicide resistance gene, heavy metal resistance gene, antibiotic resistance gene, etc.

[0053] In a specific embodiment, the resistance selection gene expression unit is 35S pro:HYG:35S term.

[0054] In some embodiments, the vector in the system is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, and a herpes simplex vector), and can also be a plasmid, a virus, a cosmid, a phage, etc., which are well known to those skilled in the art.

[0055] In one embodiment, when the target sequence is DNA, the target sequence is located at the 3' end of the protospacer adjacent motif (PAM), and the PAM has a sequence shown as TTN, wherein N is selected from A, G, T, C.

[0056] In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic cell or a eukaryotic cell.

[0057] In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.

[0058] In one embodiment, the target sequence is present in a cell. In one embodiment, the target sequence is present in the nucleus or in the cytoplasm (e.g., an organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

[0059] In one embodiment, the Cas protein is connected to one or more NLS sequences. In one embodiment, the fusion protein comprises one or more NLS sequences. In one embodiment, the NLS sequence is connected to the N-terminus or C-terminus of the protein. In one embodiment, the NLS sequence is fused to the N-terminus or C-terminus of the protein.

[0060] Protein-nucleic acid complexes / compositions

[0061] In another aspect, the present invention provides a composition (or complex) comprising:

[0062] (i) a protein component selected from: the Cas protein or the fusion protein; (ii) a nucleic acid component selected from: gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a nucleic acid encoding a precursor RNA of the gRNA; the gRNA includes a repeat sequence that binds to the Cas protein and a spacer sequence for targeting target sequence recognition, and the spacer sequence is complementary to the target sequence.

[0063] The Cas protein and gRNA of the present invention can form a binary complex, which is activated when bound to a target sequence substrate to form an activated CRISPR complex. The target sequence substrate is complementary to the spacer sequence in the gRNA (or referred to as a guide sequence that hybridizes with the target nucleic acid). In some embodiments, the spacer sequence of the gRNA completely matches the target sequence substrate. In other embodiments, the spacer sequence of the gRNA matches part (continuous or discontinuous) of the target sequence substrate, and the activated complex can exhibit side branch nuclease activity, which refers to the non-specific cutting activity or random cutting activity of the activated CRISPR complex to other nearby single-stranded nucleic acids, which is also referred to as trans cutting activity in the art.

[0064] Host cells

[0065] The present invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, wherein the cell or cell line or their progeny comprises: the Cas protein, fusion protein, polynucleotide, protein-nucleic acid composition, vector or vector system described in the present invention.

[0066] In certain embodiments, the cell is a prokaryotic cell.

[0067] In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, such as a cell of a non-human primate, a cow, a sheep, a pig, a dog, a monkey, a rabbit, a rodent (such as a rat or a mouse). In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a cell of a poultry bird (such as a chicken), a fish or a crustacean (such as a clam, a shrimp). In certain embodiments, the cell is a plant cell, such as a cell or a cultivated plant or a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, such as algae, a tree or a production plant, a fruit or a vegetable (for example, a tree such as a citrus tree, a nut tree; Solanum, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).

[0068] In certain embodiments, the cell is a stem cell or a stem cell line.

[0069] In certain cases, the host cells of the invention contain genetic or genomic modifications that are not present in their wild type.

[0070] application

[0071] The Cas protein, the fusion protein, the polynucleotide, the vector, the CRISPR-Cas system, the vector system, the composition, or the host cell provided by the present invention, in some embodiments, has any of the following uses:

[0072] (1) Applications in gene editing or gene targeting for the diagnosis and treatment of non-human diseases;

[0073] (2) Application in the preparation of gene editing or gene targeting reagents or kits;

[0074] (3) Application in gene cutting for purposes other than disease diagnosis and treatment;

[0075] (4) Application in the preparation of gene cleavage reagents or kits;

[0076] (5) Application in non-specific nucleic acid side branch cleavage;

[0077] (6) Application in non-specific nucleic acid collateral degradation;

[0078] (7) Application of gene editing in plant breeding.

[0079] Gene cleavage herein includes: DNA or RNA breakage (Cis cleavage) in the Cas protein targeting sequence described herein, breakage of non-targeted DNA or RNA (single-stranded nucleic acid substrate) in side branch cleavage (i.e., non-specific or non-targeted, Trans cleavage). In some embodiments, the cleavage is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA break or a single-stranded RNA break.

[0080] Method for constructing vector system

[0081] In the specific field of plant gene editing breeding, the present invention further constructs a vector for plant gene editing and gene targeting.

[0082] In one embodiment, the present invention provides a basic editing vector, using pCambia3301 as the vector backbone, and setting three expression units starting from the right border of T-DNA: ZmU6 pro:gRNA:AtU6-26term, ZmUBI1pro:LbCpf1:PsE9term, and 35S pro:HYG:35S term, and using the Cas protein to replace LbCpf1 to obtain the Cas-kf8 basic editing vector or the Cas-kf9 basic editing vector, respectively.

[0083] In a specific embodiment, the present invention provides a target sequence editing vector, selects gene editing sites for different regions of the target sequence to be edited, designs a recognition sequence that can pair with the editing site and the crRNA repeat sequence to form a gRNA sequence, interactively connects one or more gRNA sequences through tRNA technology, linearizes the basic editing vector, and inserts one or more gRNA sequences using DNA ligase or Gibson cloning method to form a target sequence editing vector.

[0084] As a specific embodiment, the gRNA sequence for the target sequence constructed by the present invention is shown in SEQ ID NO 13.

[0085] As a more specific example, the Cas-kf8 target sequence editing vector constructed by the present invention has a sequence as shown in SEQ ID NO 14.

[0086] Based on the above-mentioned vector, in one embodiment, the present invention provides a method for genetic transformation of plants, wherein the obtained target gene editing vector is introduced into recipient plant cells, and then heritable, non-transgenic and stably inherited offspring plants are obtained by screening.

[0087] Furthermore, the genetic transformation method comprises the following steps:

[0088] (1) genetic transformation of recipient plant cells to obtain regenerated plants (including: 1) induction of callus; 2) subculture of callus; 3) infection of callus; 4) screening and identification of resistant callus; 5) differentiation of callus into seedlings; 6) rooting culture; 7) acclimatization and transplantation to obtain regenerated plants);

[0089] (2) Detect whether the gene editing of the regenerated plants is successful;

[0090] (3) Screening to obtain homozygous edited offspring plants that have the target gene traits, are non-transgenic, and have stable inheritance.

[0091] In one embodiment, the present invention also provides a method for editing a target sequence, targeting a target sequence, or cutting a target sequence, wherein the method is for non-disease diagnosis and treatment purposes, and the subject to which the method is applied does not include humans. The method comprises contacting the target sequence with the CRISPR-Cas system, or the vector system, or the composition, or the host cell.

[0092] In one embodiment, the present invention also provides a kit for gene editing or gene targeting, which comprises the CRISPR-Cas system, or the vector system, or the composition, or the host cell.

[0093] The gene editing or editing of target nucleic acid includes modifying genes, knocking out genes, changing the expression of gene products, repairing mutations, inserting polynucleotides, and gene mutations.

[0094] In one embodiment, the present invention provides a kit for detecting a target sequence in a sample, the kit comprising:

[0095] (a) the Cas protein, or a nucleic acid encoding the Cas protein; and / or

[0096] (b) gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA comprising the gRNA, or a nucleic acid encoding the precursor RNA; the gRNA comprises a repeat sequence that binds to the Cas protein and a spacer sequence that targets the target sequence and is complementary to the target sequence; and / or

[0097] (c) a single-stranded nucleic acid detector, wherein the single-stranded nucleic acid detector is a single-stranded nucleic acid that does not hybridize with the gRNA.

[0098] In one embodiment, the present invention provides a method for detecting a target sequence in a sample, which is a method for non-disease diagnosis and treatment purposes, and comprises contacting the sample with the Cas protein, gRNA and a single-stranded nucleic acid detector, detecting a detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector, thereby detecting the target gene; the single-stranded nucleic acid detector is a single-stranded nucleic acid that does not hybridize with the gRNA.

[0099] In the present invention, the single-stranded nucleic acid detector includes but is not limited to single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifiers and other single-stranded nucleic acid detectors; "nucleic acid analogs" include but are not limited to: locked nucleic acid, bridge nucleic acid, morpholino nucleic acid, ethylene glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, arabinose nucleic acid, 2'oxymethyl RNA, 2'methoxyacetyl RNA, 2'fluoro RNA, 2'amino RNA, 4'sulfur RNA and combinations thereof, including optional ribonucleotides or deoxyribonucleotide residues.

[0100] In the present invention, the detectable signal is realized by the following means: vision-based detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, fluorescence detection, colloidal phase change / dispersion, electrochemical detection and semiconductor-based detection.

[0101] In the present invention, preferably, a fluorescent group and a quenching group are respectively arranged at both ends of the single-stranded nucleic acid detector, and a detectable fluorescent signal can be exhibited when the single-stranded nucleic acid detector is cut. The fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quenching group is selected from one or any of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

[0102] Definition of terms

[0103] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0104] The term "gRNA (guide RNA)" is any polynucleotide sequence that has sufficient complementarity with a target sequence to hybridize with the target sequence and guide the CRISPR / Cas system to specifically bind to the target sequence. The structure of gRNA usually includes a crRNA (CRISPR RNA) sequence, which is derived from the CRISPR array and contains a repeat sequence (Repeat Sequence) and a spacer sequence (Spacer Sequence). The spacer sequence is a part of foreign genetic material that complements the target sequence. Usually, gRNA can also be designed with a non-complementary single-stranded region at the 5' end called a "5' overhang", which helps to distinguish between gRNA and target sequence. In some cases, the 3' end of gRNA may contain a reverse complementary tail, which helps to improve the binding stability of gRNA and Cas protein. In some applications, gRNA may also contain additional modifications, such as phosphorylation, chemical modification, etc., to improve its stability or functionality.

[0105] The term "target sequence" has the same meaning as "target sequence" and "target nucleic acid". In the present invention, it refers to a polynucleotide targeted by the spacer sequence in crRNA. The target sequence may comprise any polynucleotide, such as DNA or RNA. The hybridization between the target sequence and crRNA will promote the expression of the CRISPR / Cas system. Complete complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the expression of a CRISPR / Cas system.

[0106] The term "non-naturally occurring" as used herein, the term "non-naturally occurring" or "engineered" are used interchangeably and represent the involvement of human effort. When these terms are used to describe nucleic acid molecules or polypeptides, they represent those nucleic acid molecules or polypeptides that do not exist in nature or are not naturally synthesized by an organism.

[0107] The term "identity" as used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or a certain position in each of the two polypeptides is occupied by lysine), then each molecule is identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have a total of 50% identity (3 out of a total of 6 positions match). Typically, the two sequences are compared when they are aligned to produce maximum identity. Such an alignment can be calculated using, for example, a computer program such as the Needleman and Wunsch (J Mol. Biol. 48:444-453 (1970)) algorithm in the GAP program, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences.

[0108] The term "plant" is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce, cabbage, cauliflower, broccoli, kale, yellow taro, melons (e.g., melon, watermelon); fruit crops, such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, blackberries, grapes, avocados, bananas , kiwi, persimmon, pomegranate, pineapple, mango, papaya, and litchi; field crops such as alfalfa, corn / maize (feed corn, sweet corn, popcorn), peanuts, small grain cereals (barley, oats, rye, rice, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oil plants (rapeseed, mustard, poppy, olive, sunflower, coconut, castor oil plant, cocoa beans, peanuts), fiber plants (cotton, flax, jute), etc.

[0109] The technical effects achieved by the present invention are:

[0110] According to the discovered and studied Cas12a effector nuclease sequence, the present invention designs, screens and performs functional verification to obtain two novel effector nucleases Cas-kf8 and Cas-kf9, which experimentally show the activity of nucleases and have broad application prospects.

[0111] The present invention obtains two new Cas12a-type effector nuclease proteins, Cas-kf8 and Cas-kf9, which have been verified by codon optimization and plant gene editing transformation experiments to have the ability to recognize and cut the target site with TTTV sequence at the 5' end, thus obtaining a new effector nuclease suitable for plant gene editing. On this basis, an efficient plant gene editing basic vector is also provided, which can effectively modify the genome of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] Figure 1 Schematic diagram of the structure of Cas-kf8 and Cas-kf9 effector nucleases and their CRISPR arrays (in the figure: (a) Cas-kf8 and Cas-kf9 are two types of V-type CRISPR-related Cas12a effector nucleases derived from Peptostreptococcus equinus and Clostridium thermobutyricum bacteria, respectively, and their respective genomes contain characteristically arranged Cas4, Cas1, and Cas2 enzyme genes and CRISPR arrays; (b) The crRNA repeat sequences of Cas-kf8 and Cas-kf9 contain the conserved sequences UCUACU and GUAGAU that can form hairpin structures at their 3' ends).

[0113] Figure 2 Schematic diagram of the structure of the rice starch synthase gene and the selected editing sites (in the figure: the starch synthase gene Waxy (LOC_Os06g04200) consists of 14 exons and 13 introns. One site in exon 6 and one site in exon 8 were selected to design OsWx-T1 and OsWx-T2 gRNAs, and PCR specific primers OsWx-F5 and OsWx-R1 were designed upstream and downstream of each for amplification and sequencing analysis of the editing region DNA fragments).

[0114] Figure 3Schematic diagram of gene editing transformation vector KF316 (in the figure: T-DNA RB is the right border of T-DNA; ZmU6pro is the promoter of maize nuclear small RNA (U6 snRNA) gene; tRNA and crRNA are the combination of tRNA and Cas12a-specific crRNA; AtU6 term is the terminator of Arabidopsis U6 snRNA gene; ZmUBI1 pro and ZmUBI1 intron are the promoters of maize ubiquitin gene including introns; Cas-kf8 is a new Cas12a effector nuclease belonging to type 2 V CRISPR system; PsE9 term is the terminator of pea ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit E9 protein gene; CaMV 35S pro is the cauliflower mosaic virus 35S promoter; HygR is hygromycin phosphotransferase; CaMV 35S term is the cauliflower mosaic virus 35S terminator; T-DNA LB is the left border of T-DNA).

[0115] Figure 4 The DNA sequence near the OsWx-T2 editing site in the target region for the Cas-kf8 gene editing event.

[0116] Figure 5 The DNA sequence of the target region of the gene editing event of Cas-kf9 (in the figure: (a) DNA sequence near the OsWx-T1 editing site; (b) DNA sequence near the OsWx-T2 editing site). DETAILED DESCRIPTION

[0117] The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and Examples, but the present invention is not therefore limited to the scope of the embodiments described. Unless otherwise specified, the experiments and methods described in the embodiments are carried out substantially according to conventional methods well known in the art and described in various references, and the reagents and raw materials used in the present invention are all commercially available. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and cannot limit the scope of the invention claimed for protection. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0118] The DNA sequence analysis and alignment, DNA vector cloning construction design, PCR primers and CRISPR gRNA design involved in the molecular biology experiments in the present invention were all completed using the DNA analysis software Geneious Prime (GraphPad Software LLC).

[0119] The concept of the present invention includes:

[0120] 1. Acquisition of novel effector nuclease genes: Based on the amino acid sequence of the Cas12a effector nuclease that has been discovered and studied, different query programs such as Blastp and Tblastn were used to search the NCBI ( www.ncbi.nlm.nih.gov ), JGI (img.jgi.doe.gov) and UniProt (www.uniprot.org) and other microbial genome and metagenomic databases, screen for recently sequenced genes with certain homology and containing the RuvC nuclease domain at the carboxyl terminus of the protein, retrieve the corresponding genomic nucleic acid sequence, use DNA analysis software such as Geneious to search for CRISPR repeat sequence arrays and protein coding regions, and identify Cas12a effector nucleases based on their conserved Cas12a-Cas4-Cas1-Cas2-CRISPR gene arrangement, preferably novel Cas12a effector nucleases with low similarity to those publicly reported, and design experiments for functional verification and screening.

[0121] 2. Verification of bacterial plasmid interference function of novel effector nuclease: Most known Cas12a effector nucleases require the target DNA sequence to be recognized to contain a conservative PAM sequence of TTTV (V is A, C, or G) at the 5' end. Based on this, a target sequence containing TTTV at the 5' end was designed, cloned into the pUC19 vector, transformed into the competent E. coli TOP10, and plasmid DNA was extracted as the target DNA. A bacterial expression unit Cas12a-gRNA containing a novel Cas12a effector nuclease and the corresponding gRNA that recognizes the above target sequence was designed, cloned into the pACYC-Duet-1 vector, transformed into Stable competent bacteria for expression, and the strain containing the Cas12a-gRNA expression vector was prepared into a new competent cell. The pUC19 plasmid DNA containing the target sequence is used to transform competent cells containing Cas12a-gRNA to verify the plasmid interference function of the new effector nuclease. If the Cas12a-gRNA has the effector nuclease function, it will recognize the target DNA sequence and cut the plasmid at a specific site, interfering with the survival of the transformed pUC19 plasmid DNA, and failing to form colonies normally. The competent control transformation without Cas12a-gRNA can form colonies normally. The difference in the number of colonies between the two reflects the strength of the plasmid interference function of the effector nuclease, and effector nucleases with stronger plasmid interference function can be screened accordingly.

[0122] 3. Establish a plant gene editing system using a novel effector nuclease: Select novel effector nucleases with strong activity in bacterial plasmid interference experiments and their corresponding crRNA repeat sequences, select suitable plant expression promoters, terminators, etc. to form gene expression units, and construct plant transformation expression basic vectors. Select rice starch synthase gene (Waxy or OsWx) LOC_Os06g04200|Chr6:1765621..1770656forward as the target gene, design two Waxy gene-specific gRNAs, clone them into the above-mentioned plant transformation vector to synchronously express the novel effector nuclease and its gRNA, and introduce Agrobacterium EHA105 for rice transformation.

[0123] 4. Plant gene editing mediated by novel effector nucleases: Use the above Agrobacterium to transform rice, select resistant callus, extract genomic DNA, PCR amplify the transformation vector-specific gene fragment, and confirm that the resistant callus is a positive transgenic event. Use the same genomic DNA to amplify the edited region of the target gene, sequence and analyze the gene editing effect, and statistically calculate the editing efficiency of different sites.

[0124] Example 1

[0125] 1. Acquisition of novel effector nuclease genes and their crRNA repeat sequences

[0126] Microbial genome query: Based on the amino acid sequences of the Cas12a effector nucleases that have been discovered and studied, such as LbCpf1 and FnCpf1 (Zetsche et al., 2015, Cell 163, 1-13), NCBI, JGI, UniProt and other microbial genome and metagenomic databases were repeatedly queried using Blastp, Tblastn, etc., and similar protein genes were obtained to remove duplicate sequences. According to the average length of known Cas12a proteins, proteins far beyond the range were removed, and only genes with complete protein lengths between 900 and 1500 amino acids were retained. Sequences that were exactly the same as known Cas12a effector nucleases were excluded, and recently sequenced genes with low similarity but containing the RuvC nuclease domain at the carboxyl terminus of the protein were screened for further screening as primary Cas12a effector nucleases.

[0127] Published patent application search: Use keywords such as Cas9, Cas12, CRISPR, etc. to search the USPTO (www.uspto.gov) and Chinese patent applications (tysf.cponline.cnipa.gov.cn), collect published effector nuclease sequences, and construct a query library. The above-mentioned preliminary selected Cas12a effector nuclease was used to search the collected effector nuclease library with Blastp, excluding sequences that are exactly the same as the known Cas12 effector nuclease, and sorted according to the degree of similarity, and those with low similarity were selected for detailed sequence analysis and selection.

[0128] Effector nuclease CRISPR sequence analysis: retrieve the genomic nucleic acid sequence corresponding to the selected Cas12a effector nuclease, use DNA analysis software Geneious to search for CRISPR repeat sequence arrays, and identify whether it is a Cas12a effector nuclease based on its conservative Cas12a-Cas4-Cas1-Cas2-CRISPR gene arrangement. Cas12a effector nucleases and their corresponding crRNA repeat sequences with complete and conservative CRISPR structures, moderate lengths, and recent sequencing are preferred, and experiments are designed for functional verification and further screening.

[0129] Through the above multi-step screening and subsequent multiple experimental verification, the inventors obtained two novel Class 2 V-type CRISPR Cas12a-like effector nucleases, named Cas-kf8 and Cas-kf9, both of which have a typical Cas12a-Cas4-Cas1-Cas2-CRISPR gene structure arrangement and CRISPR repeat sequence array ( Figure 1 a). Cas-kf8 is derived from shotgun sequencing of the Peptostreptococcus equinu genome, and its protein, nucleotide and CRISPR repeat sequences are shown in SEQ ID NOs 1-3; Cas-kf9 is derived from shotgun sequencing of the Clostridium thermobutyricum genome, and its protein, nucleotide and CRISPR repeat sequences are shown in SEQ ID NOs 4-6; wherein the two ends of the protein contain artificially added SV40 and Nucleoplasmin nuclear localization signal (NLS) sequences, respectively, and the nucleotides have been optimized to codon sequences suitable for eukaryotic cell expression; the 3' end of the crRNA repeat sequence has UCUACU and GUAGAU conservative sequences that can form a hairpin structure ( Figure 1 b).

[0130] 2. Verification of bacterial plasmid interference function of novel effector nuclease

[0131] Prokaryotic expression vectors pACYC-Duet-1 and pUC19 were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and Escherichia coli competent cells TOP10, Stable, and Agrobacterium EHA105 were purchased from Shanghai Weidi Biotechnology Co., Ltd. LB bacterial liquid medium contains 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride NaCl, and is sterilized at 121°C for 20 minutes. If antibiotics are needed, add a final concentration of 50 μg / mL after the medium is cooled moderately. LB solid medium is prepared by adding 15 g / L agar before sterilizing the LB liquid medium. Genes and primers that need to be synthesized were commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis, and DNA Sanger sequencing was completed by Sangon Biotechnology (Shanghai) Co., Ltd.

[0132] 1. Design and construction of target sequence plasmid: The fragment of rice starch synthase gene (Waxy or OsWx) LOC_Os06g04200|Chr6:1765621..1770656forward was selected as the target sequence for bacterial experiments ( Figure 2 ), two fragments located in exons 6 and 8 and containing the TTTV sequence (PAM) at their 5' ends were selected as target sequences OsWx-T1 and OsWx-T2 (SEQ ID NOs 7, 9). The same target sequences OsWx-T1 and OsWx-T2 will be tested in subsequent plant gene editing experiments.

[0133] The target recognition sequences of OsWx-T1 and OsWx-T2 (SEQ ID NO 7, 9) were artificially synthesized and cloned into pUC19 vectors to form KF280 and KF282, which were used as target genes for bacterial plasmid interference experiments. The OsWx-T1 and OsWx-T2 target sequences of KF280 and KF282 plasmid DNAs contain complete PAMs. After transformation of competent cells containing effector nuclease bacterial expression plasmids, they can be cut by the effector nuclease complex with specific recognition sequences, and the broken plasmid DNA cannot form colonies, thereby generating plasmid interference.

[0134] The target sequences of OsWx-T1 and OsWx-T2 (SEQ ID NO 8, 10) without PAM sequence were artificially synthesized and cloned into pUC19 vector to form KF281 and KF283, which were used as controls without effective target sequence for bacterial plasmid interference experiment. The incomplete OsWx-T1 and OsWx-T2 target sequences of KF281 and KF283 lack PAM. After transformation into competent cells containing bacterial expression plasmids of effector nuclease, they cannot be cut by the effector nuclease complex with specific recognition sequence. The complete plasmid DNA can form colonies normally without plasmid interference.

[0135] 2. Construction of effector nuclease bacterial expression vector:

[0136] Design and construct the effector nuclease bacterial expression vector: The prokaryotic cell expression vector pACYC-Duet-1 was used as the basic framework for modification, and the prokaryotic gene expression promoters proC and J23119 (Chavez et al., 2018, PNASUSA 115, 3669-3673; parts.igem.org / Part:BBa_J23100) were artificially synthesized to express the effector nuclease and its gRNA recognition sequence, respectively, to replace the lac repressor gene and other elements in the pACYC-Duet-1 vector, and to reserve appropriate restriction endonuclease sites for the subsequent insertion and cloning of the effector nuclease and its corresponding crRNA. LbCpf1 of Lachnospiraceaebacterium was selected as a control, and the LbCpf1 gene (SEQ ID NO 11) was artificially synthesized, and SV40 and Nucleoplasmin nuclear localization signal short peptides with 9 and 16 amino acids were added to the amino and carboxyl ends, respectively, for subsequent nuclear localization after expression in eukaryotic cells, and cloned into the proC promoter downstream of the above-mentioned improved pACYC-Duet-1 vector, and the expression was terminated by the terminator sequence of the Streptococcus pyogenes Cas9 gene. The artificially synthesized LbCpf1 crRNA+OsWx-T1 recognition sequence (SEQ ID NO 12, 8) was cloned into the J23119 promoter downstream of the above-mentioned vector, and the gene transcription was terminated by the T7 terminator downstream, and finally the bacterial expression vector KF278 of the LbCpf1 effector nuclease and the OsWx-T1 recognition sequence was formed. After OsWx-T1 in KF278 was replaced by OsWx-T2, the bacterial expression vector KF279 expressing LbCpf1 effector nuclease and OsWx-T2 recognition sequence was formed.

[0137] After replacing the LbCpf1 gene in KF278 with the Cas-kf8 or Cas-kf9 (SEQ ID NO 2, 5) gene by conventional cloning methods, bacterial expression vectors expressing the Cas-kf8 or Cas-kf9 effector nuclease and the OsWx-T1 recognition sequence are formed respectively; if the LbCpf1 gene in KF279 is replaced, bacterial expression vectors expressing the Cas-kf8 or Cas-kf9 effector nuclease and the OsWx-T2 recognition sequence are formed respectively. The bacterial expression vectors expressing the effector nuclease and the target recognition sequence are transformed into the Stable bacterial competent state for use.

[0138] 3. Bacterial plasmid interference experiment: Select Stable single colonies containing effector nuclease bacterial expression vectors KF278, KF279, etc. to culture, prepare competent cells, divide and store in a -80 degree low-temperature refrigerator, and transform with pUC19 blank vector to test the transformation efficiency to 1x10 7 cfu / μg DNA (colony forming units per microgram DNA) before plasmid interference experiments can be performed. Use 10 ng of KF280, KF281, KF282, or KF283 plasmid DNA containing the target sequence to heat shock transform competent cells containing appropriate effector nucleases and target recognition sequence expression plasmids. After 1 hour of recovery culture in liquid LB medium, appropriately dilute and plate on LB culture plates containing 50 μg / ml kanamycin. After overnight culture at 37°C, count the number of colonies. Each bacterial transformation experiment was repeated three times, and the average value was taken to calculate the transformation efficiency.

[0139] Plasmid interference experiment of control effector nuclease LbCpf1: First, use KF280, KF281, KF282, and KF283 plasmid DNA to transform blank Stable bacteria competent cells respectively, compare the number of colony formation, and adjust the concentration of each plasmid DNA to have the same transformation efficiency. Then use KF280 and KF281 plasmid DNA to transform Stable competent cells that already contain KF278 plasmid, and count the number of colonies in KF280 experiment and KF281 control. If the LbCpf1 effector nuclease and OsWx-T1 target recognition sequence expressed by KF278 can recognize and cut the OsWx-T1 target sequence in KF280, plasmid interference occurs, and the KF280 culture dish will have fewer colonies. Because the incomplete OsWx-T1 target sequence of KF281 plasmid DNA lacks the 5' end PAM sequence TTTG, it cannot be recognized and cut by the LbCpf1 effector nuclease expressed by KF278, and no plasmid interference occurs. The KF281 culture dish will have a normal number of colonies equivalent to the blank Stable competent state, which can be used to judge the enzyme cutting ability of LbCpf1 at the OsWx-T1 site in bacteria. The Stable competent state containing the KF279 plasmid is transformed with KF282 and KF283 plasmid DNA, respectively, to judge the enzyme cutting ability of LbCpf1 at the OsWx-T2 site in bacteria. Experiments have shown that LbCpf1 can effectively cut both OsWx-T1 and OsWx-T2 sites in bacteria.

[0140] Plasmid interference experiment of novel effector nuclease: Use the same KF280 and KF281 plasmid DNA as above to transform the Stable competent state containing the expression vector of novel effector nuclease Cas-kf8 or Cas-kf9 and OsWx-T1 target recognition sequence, and count the colony counts of KF280 experiment and KF281 control to determine the enzyme cutting ability of Cas-kf8 or Cas-kf9 at OsWx-T1 site in bacteria. Use KF282 and KF283 plasmid DNA to transform the Stable competent state containing the expression vector of novel effector nuclease Cas-kf8 or Cas-kf9 and OsWx-T2 target recognition sequence, and then determine the enzyme cutting ability of Cas-kf8 or Cas-kf9 at OsWx-T2 site in bacteria. The experiment proves that Cas-kf8 and Cas-kf9 have the cutting ability at both OsWx-T1 and OsWx-T2 sites in bacteria.

[0141] 3. Establishing a plant gene editing system using novel effector nucleases

[0142] Beidahuang Kenfeng Seed Industry independently developed a rice-specific gene-editing Agrobacterium transformation DNA vector, which uses the pCambia3301 vector as the backbone and contains three gene expression units ZmU6 pro:gRNA:AtU6-26 term, ZmUBI1pro:SpCas9:PsE9term, and 35S pro:HYG:35S term. A BsaI restriction site is left between ZmU6 pro and gRNA. The vector can be linearized and one or more gRNA sequences can be inserted using DNA ligase or Gibson cloning to form a basic gene editing vector for any target site. For specific sequence information, please refer to the KF80 transformation vector in patent CN113604501B "Gene editing method and application of aroma control gene in improved indica rice strains". The gene editing transformation vectors described in the present invention are all modified and constructed based on the disclosed basic gene editing vector.

[0143] The above-mentioned KF80 transformation vector was used as the basic skeleton, the SpCas9 therein was replaced by the LbCpf1 gene (SEQ ID NO 11), and the gRNA therein was replaced by an artificially synthesized tRNA-crRNA-OsWx-T1-tRNA-crRNA-OsWx-T2 (SEQ ID NO 13) fragment. The 5' and 3' ends of the fragment had 20 bp homologous to ZmU6 pro and AtU6-26 term, respectively, for Gibson cloning, and finally formed a gene editing vector KF315 based on the LbCpf1 effector nuclease, which consisted of three gene expression units ZmU6 pro:tRNA-crRNA-OsWx-T1-tRNA-crRNA-OsWx-T2:AtU6-26 term, ZmUBI1pro:LbCpf1:PsE9 term, and 35S pro:HYG:35S term, which can edit the OsWx-T1 and OsWx-T2 sites of rice. Subsequently, the LbCpf1 gene in KF315 was replaced by Cas-kf8 and Cas-kf9 (SEQ ID NO 2, 5) genes, forming gene editing vectors KF316 and KF317 based on Cas-kf8 and Cas-kf9 effector nucleases, which can also edit the OsWx-T1 and OsWx-T2 sites of rice. The composition and sequence of KF316 vector are shown in Figure 3 and SEQ ID NO 14. Except for the effector nuclease, KF315 and KF317 have the same composition structure and other sequences as KF316, so they are not listed here again. KF315, KF316, and KF317 vectors were respectively transformed into Agrobacterium EHA105 and stored at -80°C for future use.

[0144] 4. Plant gene editing mediated by novel effector nucleases

[0145] Gene editing transformation of rice: Gene editing vectors based on novel effector nucleases must be transformed into plant cells to verify their functions. Using the Agrobacterium-mediated transformation method, healthy and mature seeds of the rice variety Kendao 32 were used as explants to induce callus tissue, and the gene editing vectors KF316 and KF317 were transformed into cells, respectively, to screen for resistant transgenic events, detect whether the DNA sequences of the target genes OsWx-T1 and OsWx-T2 sites undergo specific changes, and evaluate the gene editing effects of each effector nuclease in plants. The various basic culture medium formulas used in the genetic transformation experiment are listed in Table 1, and the specific operation procedures are as follows:

[0146] Induction of rice callus: For each transformation experiment, about 200 mature, plump, healthy and clean rice seeds were selected, the husks were removed, and the seeds were placed in a 100 ml sterile glass bottle and washed with 75% ethanol for 3 times. An equal volume of sterile water and 10% sodium hypochlorite NaClO, two drops of Tween 20 were added, and the seeds were sterilized by shaking for 20 minutes, and washed with sterile water for 3-5 times until the foam was washed off. The surface moisture of the seeds was absorbed with sterile filter paper, and the seeds were placed flat on the induction medium, and cultured in a 33°C constant temperature incubator in the dark for 3-4 weeks until the callus grew out of the embryo.

[0147] Subculture of rice callus: Select and peel off the healthy callus with vigorous growth, place it on a new induction medium for subculture for 1-2 weeks, and expand and maintain the vitality of the callus.

[0148] Prepare Agrobacterium: Take out the Agrobacterium glycerol storage tube carrying the gene editing vector from the -80℃ low-temperature refrigerator, take a small amount of Agrobacterium and streak it on a solid YEP medium plate containing 50 mg / L kanamycin, and culture it in the dark at 28℃ for 2-3 days. Pick a single colony and inoculate 5 ml of YEP liquid culture medium for dark culture at 28℃ overnight. Take a small amount of bacterial liquid to extract plasmid DNA for vector-specific PCR detection to confirm that the strain carries the correct gene editing vector. At the same time, take part of the bacterial liquid to apply YEP medium plate for culture and observation. The Agrobacterium used for infection must be free of contamination by other bacteria. The whole plate must be smooth and free of granular or other colored fungi or molds to ensure the purity of the obtained Agrobacterium, and strictly aseptic operation must be performed to ensure no contamination in subsequent work. After confirming that the Agrobacterium plate is not contaminated, scrape the bacteria directly into a 100 ml Erlenmeyer flask containing 25 ml of suspension infection medium, and culture it on a rotary shaker at 100-120 rpm at a constant temperature of 25°C for 2-3 hours. Take samples to measure the OD600 value, and use the suspension infection medium to adjust the OD600 value of Agrobacterium to 0.1-0.2 before using it for callus infection.

[0149] Infection of callus tissue: Pick small granular, healthy callus tissue into a 250 ml sterile glass bottle, add Agrobacterium and shake for infection for 3-5 minutes, then use sterile filter paper to absorb the bacterial liquid on the surface of the callus tissue, place it on the sterile filter paper placed on the surface of the co-cultivation medium in advance (the filter paper prevents the growth of Agrobacterium), and co-cultivate at 28°C in the dark for 3 days.

[0150] Screening and identification of resistant callus: Transfer the infected rice callus to a sterilized 250 ml glass bottle, wash it several times with sterile water until the water is clear, and finally soak and wash it with carbenicillin at a final concentration of about 250 mg / L on a shaker at 100 rpm for 0.5 hours. Transfer the callus to a blank culture dish, dry the surface moisture of the callus with sterile filter paper, place it in a sterile clean bench and air dry it for 2.5 hours, then divide the callus and place it on the screening medium, and culture it in a 33°C constant temperature incubator in the dark for 3-4 weeks. The screening medium contains 400 mg / L carbenicillin to inhibit the growth of Agrobacterium, and 50 mg / L hygromycin to select transformed cells. Non-transformed cells stop growing and gradually die on the screening medium. After 3-4 weeks of culture, the successfully transformed cells will grow resistant callus. After the resistant callus continues to grow, appropriate amount of samples are taken to extract genomic DNA, and PCR identification is performed using primers specific to the transformation vector. Each PCR-positive callus is an independent transformation event.

[0151] Table 1 Basic culture medium composition and preparation method used in Agrobacterium genetic transformation experiment of japonica rice

[0152]

[0153]

[0154] 2. Analysis of gene editing effects:

[0155] The resistance transformation event needs to be detected by molecular biological methods to confirm whether the genetic transformation is successful. The commonly used simple and reliable detection method is PCR amplification. Sample the rice resistant callus tissue, and use a SDS (Sodium dodecylsulphate)-based DNA rapid extraction method to extract genomic DNA. First, PCR detection of the vector-specific HygR gene is performed to identify the transformation event. The PCR reaction system uses the Quick Taq HSDyeMix (DTM-101) kit (TOYOBO LifeScience). The 20-microliter PCR reaction system contains 10μl 2x Quick Taq HSDyeMix, 1.0μl 10pmol / μl primer Hyg-F1 (SEQ ID NO 15) and 1.0μl 10pmol / μl primer Hyg-R1 (SEQ ID NO 16), 6.0μl sterile water, and finally 2.0μl 50ng / μl sample genomic DNA. The PCR reaction conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute for a total of 30 cycles, and finally extension at 72°C for 7 minutes, and maintained at 4°C. The PCR amplification products were separated by 1% agarose gel electrophoresis, and samples that successfully amplified a 402 bp long fragment (SEQ ID NO17) specific to the HygR gene were determined to be transgenic positive.

[0156] By using a similar PCR amplification method and using OsWx (LOC_Os06g04200) specific primers OsWx-F5 and OsWx-R1 (SEQ ID NO 18, 19), a 916 bp target fragment (SEQ ID NO 20) near the editing site of OsWx-T1 and OsWx-T2 genes can be amplified from the HygR positive sample, and directly sequenced with the same upstream or downstream primers after purification, and compared with the same gene fragment sequence of the wild type of Kendao 32. If the DNA fragment sequence of the transgenic event is no different from the wild type sequence, gene editing has not occurred; if overlapping peaks appear near the expected effector nuclease cleavage site, it means that the sequenced PCR fragment is a mixed template, containing DNA fragments that have and have not been edited, and it is heterozygous gene editing; if there is a definite sequence difference with the wild type, it is homozygous gene editing. The gene editing experiments demonstrated for the first time that both Cas-kf8 and Cas-kf9 effector nucleases have gene editing functions, but the overall gene editing efficiency is significantly different. The editing efficiency at different sites of OsWx-T1 and OsWx-T2 is also different. The gene editing efficiency results calculated based on direct sequencing analysis of the target gene PCR fragments are shown in Table 2.

[0157] The PCR fragment of the target gene of the editing event was TOPO cloned into the pCR2.1 vector (Thermo Fisher Scientific), and 4 clones were sent for sequencing for each sample, so that the specific editing changes of the DNA sequence at the OsWx-T1 and OsWx-T2 sites could be accurately analyzed. Different clones derived from the same editing callus event often have multiple editing sequence changes. Some clones were only edited at the OsWx-T1 or OsWx-T2 sites, and some clones were edited at both sites at the same time. Therefore, the number of editing types at each site was inconsistent with the total number of editing events. Clonal sequencing further confirmed that the two new effector nucleases Cas-kf8 and Cas-kf9 both have significant gene editing activity, but there are also obvious differences between them. The editing efficiency of Cas-kf9 is about 3 times that of Cas-kf8. Cas-kf9 can edit both OsWx-T1 and OsWx-T2 sites, and there are multiple double-site edits. The editing efficiency of Cas-kf8 was relatively low. Although it failed to edit OsWx-T1, it was able to edit OsWx-T2, and the editing types were diverse (Table 2).

[0158] Table 2 Gene editing efficiency of effector nucleases

[0159]

[0160] The above target gene sequence analysis results revealed that gene editing mediated by Cas-kf8 and Cas-kf9 effector nucleases was mostly small fragment deletions, all occurring in the downstream part of the expected target gene target site, which is consistent with other known Cas12a-type effector nucleases such as LbCpf1 and FnCpf1. The detailed sequencing analysis results of each effector nuclease editing event are summarized in Table 3. The R234-xx event in the table is a callus obtained by transforming the KF316 vector containing the Cas-kf8 effector nuclease, and the R243-xx event is a callus obtained by transforming the KF317 vector containing the Cas-kf9 effector nuclease. For the convenience of description, the first T of the 5' end PAM sequence TTTV of each target site is counted as position 1. The editing sequences of some Cas-kf8 at the OsWx-T2 site and some Cas-kf9 at the OsWx-T1 and OsWx-T2 sites are shown in the table, respectively. Figure 4 and Figure 5 .

[0161] Table 3 Editing site sequence changes in OsWx gene editing events

[0162]

[0163]

[0164] The present invention obtains two new Cas12a-type effector nuclease proteins, Cas-kf8 and Cas-kf9, which have been verified by codon optimization and plant gene editing transformation experiments to have the ability to recognize and cut the target site with TTTV sequence at the 5' end, thus obtaining a new effector nuclease suitable for plant gene editing. On this basis, an efficient plant gene editing basic vector is also provided, which can effectively modify the genome of plants.

[0165] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Various modifications and changes can be made to the details according to all the teachings that have been published. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0166] Description of DNA sequences in the present invention

[0167] SEQ ID NO 1: Cas-kf8 protein 1368aa

[0168] SEQ ID NO 2:Cas-kf8 coding DNA 4104bp

[0169] SEQ ID NO 3:Cas-kf8 crRNA repeat DNA 36bp

[0170] SEQ ID NO 4: Cas-kf9 protein 1287aa

[0171] SEQ ID NO 5: Cas-kf9 coding DNA 3864bp

[0172] SEQ ID NO 6:Cas-kf9 crRNA repeat DNA 36bp

[0173] SEQ ID NO 7:synthesized OsWx-T1 fragment 29bp

[0174] SEQ ID NO 8:synthesized PAM-less OsWx-T1 fragment 25bp

[0175] SEQ ID NO 9:synthesized OsWx-T2 fragment 29bp

[0176] SEQ ID NO 10:synthesized PAM-less OsWx-T2 fragment 25bp

[0177] SEQ ID NO 11:Lachnospiraceae bacterium Cas12a(LbCpf1)CDS 3843bp

[0178] SEQ ID NO 12:Lachnospiraceae bacterium Cas12a(LbCpf1)crRNA repeat 36bp

[0179] SEQ ID NO 13:synthesized tRNA-crRNA-OsWx-T1-tRNA-crRNA-OsWx-T2 DNAfragment 286 bp

[0180] SEQ ID NO 14:KF316 DNA 10461 bp

[0181] SEQ ID NO 15:Hyg-F1 oligo 22 bp

[0182] SEQ ID NO 16:Hyg-R1 oligo 22 bp

[0183] SEQ ID NO 17:HygR gene PCR fragment 402 bp

[0184] SEQ ID NO 18:OsWx-F5 oligo 22 bp

[0185] SEQ ID NO 19:OsWx-R1 oligo 22 bp

[0186] SEQ ID NO 20:Oryza sativa Kendao32 Waxy gene OsWx(LOC_Os06g04200)PCRfragment 916 bp

Claims

1. A Cas protein, characterized in that The Cas protein is Cas-kf8 or Cas-kf9, the Cas-kf8 amino acid sequence is shown in SEQ ID No 1, and the Cas-kf9 amino acid sequence is shown in SEQ ID No 4.

2. A fusion protein comprising the Cas protein according to claim 1 and other modified parts.

3. An isolated polynucleotide, characterized in that The polynucleotide is a polynucleotide sequence encoding the Cas protein according to claim 1, or a polynucleotide sequence encoding the fusion protein according to claim 2.

4. The polynucleotide according to claim 3, characterized in that The sequence is shown as SEQ ID No 2 or 5.

5. A carrier, characterized in that The vector comprises the polynucleotide according to claim 3 or 4 and a regulatory element operably linked thereto.

6. A CRISPR-Cas system, characterized in that The system comprises the Cas protein of claim 1 and at least one gRNA, wherein the gRNA comprises a repeat sequence that binds to the Cas protein and a spacer sequence for targeting a target sequence.

7. The CRISPR-Cas system according to claim 6, characterized in that The repeating sequence is shown as SEQ ID No 3 or 6.

8. A vector system, characterized in that The vector system includes one or more vectors, which include: a gRNA expression unit and the Cas protein expression unit according to claim 1, the gRNA includes a crRNA sequence capable of targeting a target sequence, and the gRNA expression unit and the Cas protein expression unit are located on the same or different vectors of the system.

9. The carrier system according to claim 8, characterized in that The gRNA expression unit is ZmU6pro:gRNA:AtU6-26 term.

10. The carrier system according to claim 9, characterized in that A BsaI restriction site is left between ZmU6 pro and gRNA, and one or more gRNA sequences can be inserted to form a gene editing vector system targeting any target site of the target sequence.

11. The carrier system according to claim 10, characterized in that The Cas protein expression unit is ZmUBI1pro:Cas:PsE9 term.

12. The carrier system according to claim 8, characterized in that The vector system also includes a resistance selection gene expression unit.

13. The carrier system according to claim 12, characterized in that The resistance screening gene expression unit is 35Spro:HYG:35S term.

14. A composition, characterized in that The composition comprises: (i) a protein component selected from: the Cas protein of claim 1 or the fusion protein of claim 2; (ii) a nucleic acid component selected from: gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a nucleic acid encoding the precursor RNA of the gRNA; the gRNA comprises a repeat sequence that binds to the Cas protein and a spacer sequence for targeting a target sequence.

15. An engineered host cell, characterized in that The host cell comprises the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3 or 4, or the vector of claim 5, or the CRISPR-Cas system of claim 6 or 7, or the vector system of any one of claims 8 to 13, or the composition of claim 14.

16. Use of the Cas protein according to claim 1, or the fusion protein according to claim 2, or the polynucleotide according to claim 3 or 4, or the vector according to claim 5, or the CRISPR-Cas system according to claim 6 or 7, or the vector system according to any one of claims 8 to 13, or the composition according to claim 14, or the host cell according to claim 15 in any of the following: (1) Applications in gene editing or gene targeting for the diagnosis and treatment of non-human diseases; (2) Application in the preparation of gene editing or gene targeting reagents or kits; (3) Application in gene cutting for purposes other than disease diagnosis and treatment; (4) Application in the preparation of gene cleavage reagents or kits; (5) Application in non-specific cleavage of side branch nucleic acids; (6) Application in non-specific degradation of collateral nucleic acids; (7) Application of gene editing in plant breeding.

17. A basic editing vector, characterized in that Using pCambia3301 as the vector backbone, three expression units were set starting from the right border of T-DNA: ZmU6 pro:gRNA:AtU6-26term, ZmUBI1 pro:LbCpf1:PsE9term, and 35Spro:HYG:35S term, and the Cas protein described in claim 1 was used to replace LbCpf1 to obtain a Cas-kf8 basic editing vector or a Cas-kf9 basic editing vector, respectively.

18. A target sequence editing vector, characterized in that: Design recognition sequences for different sites of the target sequence to be edited, form a gRNA sequence with the appropriate crRNA repeat sequence, interactively connect one or more gRNA sequences through tRNA technology, linearize the basic editing vector described in claim 17, and insert one or more gRNA sequences using DNA ligase or Gibson cloning method to form a target sequence editing vector.

19. A method for genetic transformation of a plant, characterized in that: The target sequence editing vector of claim 18 is introduced into a recipient plant cell, and then screened to obtain heritable, non-transgenic and stably inherited offspring plants, preferably comprising the following steps: (1) Genetically transforming recipient plant cells to obtain regenerated plants; (2) Detecting whether gene editing of the regenerated plants is successful; (3) Screening to obtain offspring plants that are homozygous for the target sequence traits, non-transgenic, and stably inherited.

20. A method for editing a target sequence or targeting a target sequence, characterized in that: The method is for non-disease diagnosis and treatment purposes, the subject to which the method is applied does not include humans, and the method comprises contacting the target sequence with the CRISPR-Cas system of claim 6 or 7, or the vector system of claim 8, or the composition of claim 14, or the host cell of claim 15.

21. A method for cutting a target sequence, characterized in that: The method is for non-disease diagnosis and treatment purposes, the subject to which the method is applied does not include humans, and the method comprises contacting the target sequence with the CRISPR-Cas system of claim 6 or 7, or the vector system of claim 8, or the composition of claim 14, or the host cell of claim 15.

22. A kit for gene editing or gene targeting, characterized in that: The kit comprises the CRISPR-Cas system of claim 6 or 7, or the vector system of any one of claims 8 to 13, or the composition of claim 14, or the host cell of claim 15.

23. A kit for detecting a target sequence in a sample, characterized in that: The kit comprises: (a) the Cas protein of claim 1, or a nucleic acid encoding the Cas protein; and / or (b) gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA comprising the gRNA, or a nucleic acid encoding the precursor RNA; the gRNA comprises a crRNA sequence capable of targeting a target sequence; and / or (c) a single-stranded nucleic acid detector, wherein the single-stranded nucleic acid detector is a single-stranded nucleic acid that does not hybridize with the gRNA.

24. A method for detecting a target sequence in a sample, characterized in that: The method is for non-disease diagnosis and treatment purposes, and comprises contacting a sample with the Cas protein, gRNA and single-stranded nucleic acid detector described in claim 23, detecting a detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector, thereby detecting a target sequence; the single-stranded nucleic acid detector is a single-stranded nucleic acid that does not hybridize with the gRNA.

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