RU2771626C1 - Tool for cutting double-stranded dna using cas12d protein from katanobacteria and hybrid rna produced by fusion of guide crispr rna and scout rna - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular, to hybrid RNA (sgRNA) used as guide RNA in the Cas12d system for editing genomic DNA, as well as to a DNA cassette for expression thereof.

EFFECT: invention is effective for changing the sequence of genomic DNA of a unicellular or multicellular organism.

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Description

Technical field

The invention relates to the field of molecular biology and microbiology, in particular, describes new bacterial nucleases of the CRISPR-Cas system and new hybrid forms of CRISPR-RNA guides. The invention can be used as a tool for highly specific DNA modification in various organisms.

The invention was created with the financial support of the Ministry of Science and Higher Education of the Russian Federation under Agreement No. 075-15-2019-1661 dated 10/31/2019.

State of the art

Changing the DNA sequence is one of the urgent tasks of biotechnology today. Editing and modifying the genomes of eukaryotic and prokaryotic organisms, as well as manipulations with DNA in vitro, require the targeted introduction of double-strand breaks in the DNA sequence. To solve this problem, the following methods are currently used: artificial nuclease systems containing zinc finger domains, TALEN systems, and bacterial CRISPR-Cas systems. The first two methods require labor-intensive optimization of the nuclease amino acid sequence to recognize a particular DNA sequence. In contrast to them, in the case of CRISPR-Cas systems, the structures that recognize the target DNA are not proteins, but short guide RNAs. Cutting a specific target DNA does not require de novo synthesis of the nuclease or its gene, but is achieved through the use of guide RNAs complementary to the target sequence. This makes CRISPR-Cas systems convenient and efficient tools for cutting various DNA sequences. The technique allows simultaneous cutting of DNA in several regions using guide RNAs of different sequences. This approach is used, among other things, to simultaneously change several genes in eukaryotic organisms.

By their nature, CRISPR-Cas systems are the immune systems of prokaryotes capable of highly specific ruptures in the genetic material of viruses (Mojica et al., 2005). The abbreviation CRISPR-Cas stands for “Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated genes” (Jansen et al., 2002). All CRISPR-Cas systems consist of CRISPR cassettes and genes encoding various Cas proteins (Jansen, 2002). CRISPR cassettes consist of spacer sequences, each with a unique nucleotide sequence, and repeated palindromic repeats (Jansen, 2002). As a result of transcription of CRISPR cassettes and their subsequent processing, guide crRNAs are formed, which, together with Cas proteins, form an effector complex (Brouns et al., 2008). By complementary pairing of crRNA with a target DNA region, called a protospacer, Cas nuclease recognizes the target DNA and introduces a break in it in a highly specific manner.

CRISPR-Cas systems represented by a single effector protein are divided into six different types (from I to VI) depending on the Cas proteins that make up the systems.

The CRISPR-Cas12d system belongs to type V and is characterized by a simple composition and mechanism of operation: its functioning requires the formation of an effector complex consisting of only one Cas12d protein and two short RNAs: crRNA (crRNA) and scout RNA (scoutRNA). The scout RNA pairs complementarily with the crRNA region derived from the CRISPR repeat, forming a secondary structure necessary for binding guide RNAs to the Cas effector. Despite some similarities, scout RNA and its binding mechanism to crRNA are very different from tracer RNA, which is characteristic of Cas9 proteins, in particular SpCas9 from Streptococcus pyogenes, a nuclease widely used in biotechnology.

The effector protein Cas12d is an RNA-dependent DNA endonuclease that forms double-strand breaks in DNA (Harrington et al., 2020).

The use of CRISPR-Cas nucleases to modify the genomes of various organisms requires recreating their activity in vitro and creating a hybrid form of guide RNA (sgRNA), where crRNA and a second, additional RNA are fused into a single molecule. Such molecules were created for SpCas9 and improve the usability of the system by reducing the number of its components and reducing the number of promoters required for gene expression in various organisms. However, for CRISPR-Cas12d systems, such hybrid RNAs have not been created previously.

To date, there are several CRISPR-Cas nucleases, whose work has been reconstructed in vitro, capable of directing and specifically introducing double-strand breaks in DNA, the most popular of which is SpCas9 from Streptococcus pyogenes. One of the main characteristics limiting the use of CRISPR-Cas systems is the PAM sequence that flanks the target DNA from the 3'-end, the presence of which is necessary for the correct recognition of Cas DNA by nuclease. Different CRISPR-Cas proteins have different PAM sequences, which limit the application of nucleases to any DNA region. Thus, SpCas9 requires 5'-NGG-3' PAM.

The use of CRISPR-Cas proteins with new diverse PAM sequences is necessary to ensure the possibility of changing any DNA region, both in vitro and in the genome of living organisms. Altering eukaryotic genomes also requires the use of small nucleases to enable delivery of CRISPR-Cas systems to cells via AAV viruses. Cas12d nucleases, which are small in size, can solve the problem of delivery of CRISPR-Cas systems in viral capsids of limited capacity.

Despite the popularity of a number of methods for cutting DNA and changing the sequence of genomic DNA, today there is a need for new effective tools for modifying DNA in various organisms and at strictly defined places in the DNA sequence.

This invention has a number of properties necessary to solve this problem.

The essence of the invention

The objective of the present invention is to create new tools for changing the sequence of genomic DNA of unicellular or multicellular organisms based on CRISPR-Cas12d systems.

The current CRISPR-Cas systems are of limited use due to the specific PAM sequence that must be present at the 3' end of the DNA region being modified. The search for new Cas enzymes with other PAM sequences will expand the arsenal of available tools for the formation of a double-strand break in the necessary, strictly defined places in the DNA molecules of different organisms. The search for new Cas enzymes of small size will allow their delivery in AAV vectors.

Cas12d proteins have a small size and short PAM sequences, which are often found in genomes, which expands their application in biotechnology. However, to date, only one Cas12d nuclease from termite symbiont bacteria has been reconstructed in vitro. In addition, no hybrid guide RNA (sgRNA) has been created for any member of the Cas12d family, which, similarly to sgRNA used for Cas9 proteins, would reduce the number of system components and facilitate their expression in the cells of organisms.

The task of the work is to create a DNA cutting tool based on Cas12d proteins and hybrid RNA obtained by fusion of CRISPR guide RNA and scout RNA.

To solve this problem, the authors reconstructed in vitro the nuclease activity of the CRISPR-Cas12d system from Katanobacteria (KbCas12d), whose activity was previously shown only in bacterial cells (US2020255858A1), and for the first time for systems of the Cas12d type, a new type of hybrid RNA was created, which is a fusion of the guide CRISPR RNA and scout RNA.

CRISPR-KbCas12d with guide fusion RNA can be used to introduce targeted changes in the genome of this and other organisms. The essential features that distinguish the present invention are: (a) the possibility of using one guide RNA (sgRNA) instead of two (crRNA and scoutRNA); (b) the small size of the characterized KbCas12d protein - 1125 amino acid residues (a.a.), which is 243 a.a. less than the size of the known Cas9 enzyme from Streptococcus pyogenes (SpCas9); (c) a wide operating temperature range of the KbCas12d nuclease, which is active at temperatures from 25°C to 45°C with an optimum at 35°C, which will allow its use in organisms with different temperatures.

This problem is solved by using a protein having a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, to form a double-strand break in the DNA molecule, located immediately after the 5'-TA-3' nucleotide sequence in the specified DNA molecule . In some embodiments of the invention, this application is characterized in that the formation of a double-strand break in the DNA molecule occurs at a temperature of from 25°C to 45°C.

This problem is also solved by creating a method for changing the genomic DNA sequence of a unicellular or multicellular organism, including introducing into at least one cell of this organism an effective amount of: a) either a protein having a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO : 1, or a nucleic acid encoding a protein gene having a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, and b) a guide hybrid RNA containing a sequence that forms a duplex with the nucleotide sequence of a portion of the genomic DNA of the organism, immediately adjacent to the nucleotide sequence 5'-TA -3', and interacting with the specified protein after duplex formation, while the interaction of the specified protein with the guide RNA and the nucleotide sequence 5'-TA-3' leads to the formation of a double-strand break in the genomic sequence th DNA immediately adjacent to the 5'-TA-3' sequence. In some embodiments of the invention, this method is characterized by further comprising the introduction of an exogenous DNA sequence simultaneously with the guide RNA.

As a guide RNA, a mixture of crRNA (crRNA) and scout RNA (scoutRNA) can be used, capable of forming a complex with the target DNA site and the KbCas12d protein. In preferred embodiments of the invention, a hybrid RNA developed by the authors and constructed on the basis of crRNA and scout RNA by merging their nucleotide sequences with the general formula A-B-C-D can be used as a guide RNA, where A is the sequence of KbCas12d scout RNA, B is linker sequence; C - sequence of the direct repeat DR KbCas12d crRNA, D - sequence complementary to the target DNA (spacer segment); in this case, the sequence of the linker and the spacer segment can be any.

In some embodiments, the sequence of the KbCas12d scout RNA is synthesized based on the sequence of SEQ ID NO: 6. In some embodiments, the direct repeat DR sequence of the KbCas12d crRNA is an invariant part of the sequence of SEQ ID NO: 3.

The fusion RNA, in turn, can be produced using a fusion RNA expression cassette and consists of a U6 promoter sequence, a fusion RNA sequence according to claim 1, and a DNA sequence flanked at the 5' end of the PAM by a 5'-TA-3' sequence.

The invention can be used both for cutting the target DNA in vitro and for modifying the genome of any living organism. Genome modification can be carried out in a direct way - by cutting the genome at the appropriate site, as well as by inserting an exogenous DNA sequence due to homologous repair.

Any section of double-stranded or single-stranded DNA from the genome of an organism other than the organism used for administration (or a mixture of such sections among themselves and with other DNA fragments) can be used as an exogenous DNA sequence, while this section (or mixture of sections) is intended for integration into the site of a double-strand break in the target DNA formed under the action of the KbCas12d nuclease. In some embodiments of the invention, a portion of double-stranded DNA from the genome of an organism used when introducing the KbCas12d protein, but altered by mutations (substitution of nucleotides), as well as insertions or deletions of one or more nucleotides, can be used as an exogenous DNA sequence.

The technical result of the present invention is to increase the versatility of the available CRISPR-Cas12d systems, allowing the use of the Cas12d nuclease to cut genomic or plasmid DNA at more specific sites and at larger temperature ranges. Another technical result of the present invention is the simplification of genome editing by systems of the Cas12d family through the use of a hybrid guide RNA.

Brief description of the drawings

Fig. 1. Scheme of the CRISPR-KbCas12d locus.

Fig. 2. KbCas12d in complex with scout RNA and crRNA cuts the target DNA in vitro.

Fig. Fig. 3. Activity of the KbCas12d protein in DNA cutting in vitro depending on temperature.

Fig. Fig. 4. DNA cutting with KbCas12d protein in complex with crRNA and different forms of scout RNA shown in the figure above.

Fig. 5. Creation of hybrid RNA for KbCas12d nuclease. (A) sgRNA fusion design scheme. (B) The result of sgRNA DNA cutting with forms having linker sequences of different lengths to connect crRNA and scout RNA.

Fig. 6. Cutting KbCas12d various target DNAs in vitro.

Fig. 7. Alignment of the amino acid sequences of KbCas12d and Cas12d15 from termite symbiont bacteria using the NCBI BLASTp program (default parameters).

Detailed disclosure of the invention

In the description of the present invention, the terms "comprises" and "comprising" are interpreted to mean "includes, among other things." These terms are not intended to be construed as "consisting only of". Unless otherwise defined, the technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

As used herein, the term "percent homology of two sequences" is equivalent to the term "percent identity of two sequences". Sequence identity is determined based on the reference sequence. Algorithms for sequence analysis are known in the art, such as BLAST as described by Altschul et al. in 1990. For the purposes of the present invention, comparison of nucleotide and amino acid sequences can be used to determine the level of identity and similarity between nucleotide sequences and amino acid sequences using the Blast software package provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih .gov/blast) using a broken alignment with default settings. The percent identity of two sequences is determined by the number of positions of identical amino acids in these two sequences, taking into account the number of gaps and the length of each gap, which must be entered for optimal matching of two sequences by alignment. The percent identity is equal to the number of identical amino acids at given positions, taking into account the alignment of the sequences, divided by the total number of positions and multiplied by 100.

The term "specifically hybridizes" refers to an association between two single-stranded nucleic acid molecules, or sufficiently complementary sequences, to permit such hybridization under predetermined conditions commonly used in the art.

The phrase "double-strand break located immediately after the PAM nucleotide sequence" means that a double-strand break in the target DNA sequence will be made at a distance of 0 to 25 nucleotides after the PAM nucleotide sequence.

An exogenous DNA sequence administered simultaneously with a guide RNA is to be understood as a DNA sequence prepared specifically for the specific modification of a double-stranded target DNA at the break site determined by the specificity of the guide RNA. Such a modification may be, for example, the insertion or deletion of certain nucleotides at the site of a break in the target DNA. Exogenous DNA can be either a stretch of DNA from another organism or a stretch of DNA from the same organism as the target DNA.

A protein containing a specific amino acid sequence is to be understood as a protein having an amino acid sequence composed of the specified amino acid sequence and possibly other sequences connected by peptide bonds to the specified amino acid sequence. Other sequences are exemplified by the nuclear localization signal (NLS) sequence, or other sequences that provide increased functionality for the specified amino acid sequence.

An exogenous DNA sequence administered simultaneously with a guide RNA is to be understood as a DNA sequence prepared specifically for the specific modification of a double-stranded target DNA at the break site determined by the specificity of the guide RNA. Such a modification may be, for example, the insertion or deletion of certain nucleotides at the site of a break in the target DNA. Exogenous DNA can be either a stretch of DNA from another organism or a stretch of DNA from the same organism as the target DNA.

An effective amount of protein and RNA introduced into a cell should be understood as such an amount of protein and RNA that, when it enters the specified cell, will be able to form a functional complex, that is, a complex that will specifically bind to the target DNA and produce a double-strand break in it at a location determined by guide RNA and PAM sequence on DNA. The efficiency of this process can be assessed by analyzing the target DNA isolated from said cell using standard methods known to those skilled in the art.

Delivery of protein and RNA into the cell can be carried out in various ways. For example, a protein can be delivered as a DNA plasmid that encodes the gene for that protein, as an mRNA for translation of that protein in the cell's cytoplasm, or as a ribonucleoprotein complex that includes the protein and a guide RNA. Delivery can be accomplished by various methods known to those skilled in the art.

The nucleic acid encoding the components of the system can be introduced into the cell directly or indirectly: by transfection or transformation of cells by methods known to those skilled in the art, by the use of a recombinant virus, by cell manipulation such as DNA microinjection, and the like.

Delivery of a ribonucleic complex consisting of a nuclease and guide RNAs and exogenous DNA (if necessary) can be carried out by transfection of the complexes into the cell or by mechanical introduction of the complex into the cell, for example, microinjection.

The nucleic acid molecule encoding the protein to be introduced into the cell may be integrated into a chromosome or may be extrachromosomally replicating DNA. In some embodiments, to ensure efficient expression of a protein gene with DNA introduced into a cell, it is necessary to change the sequence of this DNA in accordance with the cell type in order to optimize codons during expression, due to the uneven frequency of occurrence of synonymous codons in the coding regions of the genome of various organisms. Codon optimization is required to increase expression in animal, plant, fungal, or microbial cells.

For a protein having a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 to function in a eukaryotic cell, the protein must be in the nucleus of that cell. Therefore, in some embodiments of the invention, a protein is used to form double-strand breaks in the target DNA, having a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, and which is further modified at one or both ends by the addition of one or more nuclear localization signals. For example, a nuclear localization signal from the SV40 virus can be used. For efficient delivery to the nucleus, the nuclear localization signal can be separated from the main protein sequence by a spacer sequence, such as that described by Shen et al. in 2013. Also, in other embodiments, a different nuclear localization signal, or alternative method of delivering said protein to the cell nucleus, can be used.

The present invention encompasses the use of a protein from the organism Katanobateria homologous to the previously characterized in vitro protein Cas12d15 from termite symbiote bacteria to introduce double-strand breaks in DNA molecules at well-defined positions.

The use of CRISPR nucleases to introduce targeted changes in the genome has a number of advantages. First, the specificity of the system's action is determined by the crRNA sequence, which makes it possible to use one type of nuclease for all target loci. Secondly, the technique allows several guide RNAs, complementary to different target genes, to be delivered into the cell at once, which makes it possible to carry out a one-time change in several genes at once.

KbCas9 - Cas nuclease found in Katanobacteria. The Katanobacteria CRISPR Cas12d1 system (hereinafter CRISPR KbCas12d) belongs to the V D type of CRISPR Cas systems and consists of a CRISPR cassette carrying direct repeats (DR) with a 5' actccgaaagtatcggggataaaggc 3' sequence separated by unique spacer sequences. The activity of the CRISPR-KbCas12d system as a protective system was previously shown in bacteria (Harrington et al., 2020), during the same experiments, the requirements for PAM were determined, but in vitro the operation of the system was not previously recreated. The CRISPR cassette is adjacent to the gene for the effector Cas12d protein KbCas12d, as well as the gene for the Cas1 protein involved in adaptation and incorporation of new spacers. Next to the Cas genes is a sequence partially complementary to direct repeats, folding into a characteristic secondary structure, the putative scout RNA (scoutRNA) (Fig. 1). However, the functioning of scout RNA in KbCas12d has not been experimentally shown.

Reconstruction of the DNA-cutting complex KbCas12d in vitro

Previous bioinformatic analysis (Harrington et al., 2020) predicted putative crRNA and scout RNA sequences for the CRISPR-KbCas12d system (Table 1).

Table 1. CRISPR-KbCas12d guide RNA sequences used for primary tests of KbCas12d nuclease activity in vitro. Bold indicates the DR direct repeat sequence.

Name Subsequence KbCas12d scout RNA 5'
SEQ ID NO: 2 KbCas12d crRNA 5'-acuccgaaaguaucggggauaaaggcnnnnnnnnnnnnnnnnnn-3'
(SEQ ID NO: 3)

To test the activity of KbCas12d nuclease, experiments were carried out to recreate the DNA cutting reaction in vitro. To do this, it was necessary to obtain all components of the KbCas12d effector complex: guide RNA and nuclease in recombinant form. Sequencing of guide RNAs made it possible to synthesize crRNA and scout RNA molecules in vitro. Synthesis was performed using the NEB HiScribe T7 RNA synthesis kit.

A linear DNA fragment 916 bp long was used as a target DNA. (SEQ ID NO: 4). The target DNA carried the target site 5' GTCATTGGCAGCTACAGG 3' flanked from the 5' end of the PAM by the sequence 5' TA 3'

To cut this target, guide RNAs of the following sequence were used: scout RNA (SEQ ID NO: 2; 5' aaguaucaaaauaaaaaggguuuccaguuuuuaacuaaacuuuuagccuuccacccuuuc -3') and crRNA (SEQ ID NO: 5; 5' cuccgaaaguaucgggauaaaggcGUCAUUGGCAGCUACAGG 3'). The crRNA sequence complementary to the protospacer (target DNA sequence) is highlighted in bold.

To obtain the recombinant KbCas12d protein, its gene was cloned into the pET21a plasmid together with the MBP (Maltose Binding Protein) coding sequence. As DNA encoding the gene, DNA with a sequence matching the corresponding gene in the genome of the host bacterium Katanobacteria was used. Escherichia coli Rosetta cells were transformed with the obtained plasmid pET21a-MBP-KbCas12d-6xHis.

5 ml of the overnight culture was diluted in 500 ml of LB medium, and the cells were grown at a temperature of 37°C until an optical density of 0.6 relative units was reached. Target protein synthesis was induced by adding IPTG to a concentration of 1 mM, after which the cells were incubated at 16°C for 16 hours. Then the cells were centrifuged at a speed of 5000 g for 30 minutes, the resulting cell pellets were frozen at -20°C.

The precipitates were thawed on ice for 30 minutes, resuspended in 15 ml of lysis buffer (HEPES 50mM pH 7.5 (24°C), 10% glycerol, 500 mM NaCl, 10 mM imidazole, 1 mM beta-mercaptoethanol) with the addition of 15 mg lysozyme and again incubated on ice for 30 minutes. The cells were then disrupted by sonication for 30 minutes and centrifuged for 40 minutes at 16,000 g. The resulting supernatant was passed through a 0.2 µm filter and applied to a HisTrap HP 1 mL column (GE Healthcare) at a rate of 0.4 mL/min.

Chromatography was performed using an AKTA FPLC chromatograph (GE Healthcare) at a speed of 0.4 ml/min. The protein loaded column was washed with 20 ml of lysis buffer with the addition of 30 mM imidazole, after which the protein was washed with lysis buffer with the addition of 300 mM imidazole.

Next, the protein was concentrated using an Amicon concentrator (with a 30 kDa filter) to a volume of 500 µl in a buffer containing no imidazole. TEV protease (30 μl TEV from a 2 mg/ml stock) was then added to the protein to cut off the MBP tag and incubated overnight at +4°C.

Then, the protein fraction obtained during affinity chromatography was passed through a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare) equilibrated with a buffer containing 50 mM Hepes-HCl pH=7.5, 500 mM NaCl, 1 mM DTT and 10 % glycerin. Using an Amicon concentrator (with a 30 kDa filter), the fractions corresponding to the monomeric form of the KbCas12d protein were concentrated to 1 mg/ml, after which the purified protein was stored at -80°C in ^a buffer containing 10% glycerol.

In vitro linear DNA cutting reaction was carried out in a volume of 20 µl under the following conditions. The reaction mixture consisted of: 1X CutSmart Buffer (NEB), 20 nM DNA, 4 µM scout RNA/crRNA, 400 nM KbCas12d protein. Samples containing no scoutRNA were prepared similarly as controls. Samples were incubated at 37°C and analyzed by gel electrophoresis in 1.5% agarose gel. In the case of correct recognition and specific cutting of DNA by the KbCas12d protein, two DNA fragments of the order of 670 and 246 base pairs in length should be formed (see Fig. 2).

The experimental results showed that the reconstructed KbCas12d-crRNA-scoutRNA complex is active in vitro and cuts DNA carrying the target protospacer sequence.

The temperature gradient (Fig. 3) showed that the protein is active in the temperature range of 25°C to 45°C. Later in the work, a temperature of 37°C was used as a working temperature.

Design of a hybrid RNA obtained by fusion of CRISPR guide RNA and scout RNA.

Next, the selection of the optimal sequence of scout RNA was carried out. For this, a number of forms of scout RNA were synthesized in vitro, differing in length and secondary structure (Fig. 4):

Scout RNA 1 aaaggguuuccaguuuuuaacuaaaacuuuagccuuccacccuuuu (SEQ ID NO: 6);

Scout RNA 2 caaaauaaaaaggguuuccaguuuuuaacuaaacuuuuagccuuccacccuuuccugauuuug (SEQ ID NO: 7);

Scout RNA 3

aaguaucaaaauaaaaaggguuuccaguuuuuaacuaaacuuuagccuuccacccuuuccugauuuuguugauaau

(SEQ ID NO: 8);

Scout RNA 4 aaguaucaaaauaaaaaggguuuccaguuuuuaacuaaacuuuagccuuccacccuuuc (SEQ ID NO: 9);

Scout RNA 5 auuaucaacaaaaucaggaaaggguggaaggcuaaaguuuaguuaaaaacuggaaacccuuu (SEQ ID NO: 10).

With synthesized scout RNAs, crRNA sequence SEQ ID NO: 5 and recombinant protein KbCas12d, a target DNA cutting reaction (SEQ ID NO: 4) was carried out in a volume of 20 μl at 37°C for 30 minutes (1X CutSmart buffer (NEB), 20 nM DNA, 4 μM scoutRNA/crRNA, 400 nM KbCas12d protein) (Figure 4)

The results of the experiment showed that scout RNA1 (aaaggguuuccaguuuuuaacuaaacuuuuagccuuccacccuuu, SEQ ID NO: 6), the shortest form of the molecule, is optimal for DNA cutting. In complex with it and crRNA, the KbCas12d protein actively cuts a double-stranded DNA fragment carrying a protospacer sequence flanked by PAM 5' TA 3'.

This molecule was used as the basis for the design of a hybrid RNA fusion guide crRNA and scout RNA (sgRNA, single guide RNA, sgRNA) (FIG. 5). To create sgRNA, the 3' end of the scout RNA was connected to the crRNA sequence via linkers with the “GAAA” sequence. sgRNAs with different linker lengths were tested (Figure 5):

sgRNA 1 (SEQ ID NO: 11) (aaaggguuuccaguuuuuaacuaaacuuuagccuuccacccuuugaaacuccgaaaguaucggggauaaaggcAUCAAUACCAAACUCUGG)

sgRNA 2 (SEQ ID NO: 12)

(aaaggguuuccaguuuuuaacuaaacuuuagccuuccacccuuugaaagaaacccgaaaguaucggggauaaaggcAUCAAUACCAAACUCUGG)

sgRNA 3 (SEQ ID NO: 13)

(aaaggguuuccaguuuuuaacuaaacuuuagccuuccacccuuugaaagaaagaaacccgaaaguaucggggauaaaggcAUCAAUACCAAACUCUGG)

The sgRNA sequence that recognizes the protospacer is in bold; the linker sequence is shown in bold-italics.

Protein KbCas12d in complex with sgRNA 1, sgRNA 2 or sgRNA 3 was incubated with the DNA target used in the above experiments for 30 minutes at 37°C. The reaction products were applied to agarose electrophoresis to assess the efficiency of cutting the DNA fragment. A reaction mixture was used as a positive control, where a complex of crRNA and scout RNA was used instead of sgRNA.

The result of the experiment showed that the use of sgRNA 1, sgRNA 2 lead to effective cutting of the target DNA, along with a set of guide RNAs of two types - scout and crRNA. Thus, it was possible to find a form of hybrid RNA (sgRNA) for effective DNA cutting by the KbCas12d protein. These fusion RNA variants can be used to cut any other target DNA by changing the sequence that directly pairs with the target DNA.

A similar scheme for connecting scout RNA and crRNA can be used for any member of the Cas12d family.

The following examples of the implementation of the method are given in order to disclose the characteristics of the present invention and should not be construed as in any way limiting the scope of the invention.

Example 1 Testing the activity of the KbCas12d protein in cutting various target DNAs.

In order to test the ability of KbCas12d to recognize different DNA sequences flanked by the PAM sequence, in vitro cutting experiments on DNA targets from the human grin2b gene sequence were performed (see Table 2). The target DNA was a PCR product matching the sequence of the human gene fragment grin2b: SEQ ID NO: 14.

As a negative control, a target DNA flanked at the 5' end with nucleotides other than PAM “TA” was cut.

In the cutting reaction, a PCR fragment of the grin2b gene carrying recognition sites (Table 2) presumably recognized by KbCas12d was used as a target. To recognize these sequences, we synthesized crRNAs that direct KbCas12d to these sites.

Table 2. DNA targets of the human grin2b gene.

5' flanking region (PAM in bold) DNA target Target1 ATTCTCTGGTA TTTGCTCTGCAGAATGAG Target 2 AAGGACCTTTA TCTCCTTTCATTGAGCAC Target 3 GAGCATGTTA AAATAGGATCTACATCAC Target 4 ACCCGGGGTA CCACGGAGAGATGGTGGA Target 5 TATTGCTATA GTCATTGGCAGCTACAGG Target 6 TTGGCAGCTA CAGGCAGAGACAAAGGAG Target 7 (without PAM) GCCATCCTAT AGTCGTGACTTCCCTAAA

The cutting reactions were carried out under conditions adjusted for KbCas12d, the result is shown in FIG. 6. From FIG. 6 it can be seen that the KbCas12d enzyme successfully cut all targets with the appropriate PAM and did not introduce breaks in target 7 used as a negative control.

Thus, the conducted research tests made it possible to restore the activity of the CRISPR-KdCas12d system from Katanobacteria in vitro, to create a DNA cutter based on it, consisting of a hybrid RNA KbCas12d nuclease obtained by fusion of CRISPR guide RNA and scout RNA.

Example 3 Cas12d proteins from closely related organisms belonging to Katanobacteria.

To date, for CRISPR-Cas12d family systems, only the activity of the CRISPR-Cas12d15 system from termite symbionts has been reconstructed in vitro (Harrington et al., 2020). Cas12d15 has a size comparable to KbCas12d and is 27% identical to KbCas12d (Fig. 7, the degree of identity was calculated using the BLASTp program, default parameters).

Thus, the KbCas12d protein significantly differs in amino acid sequence from other Cas12d proteins whose activity has been restored in vitro. KbCas12d is the first Cas12d protein for which the possibility of creating a hybrid sgRNA has been shown.

It is obvious to a specialist in the field of genetic engineering that the variant of the KbCas12d protein sequence obtained and characterized in this Description can be changed without changing the function of the protein itself (for example, by directed mutagenesis of amino acid residues that do not directly affect functional activity (Sambrook et al., 1989). in particular, the person skilled in the art knows that non-conservative amino acid residues can be changed that do not affect the residues that determine the functionality of the protein (defining its function or structure. Examples of such changes can be the replacement of non-conservative amino acid residues with homologous ones. In some embodiments of the invention, it is possible to use a protein containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 and differs from SEQ ID NO: 1 only in non-conservative amino acid residues, to form a double-strand break in the mole a DNA stub located immediately after the 5'-TA-3' nucleotide sequence in said DNA molecule. Homologous proteins can be obtained by mutagenesis (eg, site-directed or PCR-mediated mutagenesis) of the appropriate nucleic acid molecules, followed by testing the encoded modified Cas12d protein for retention of its functions in accordance with the functional assays described here.

Example 3. The KbCas12d system described in the present invention in combination with guide RNAs or the hybrid RNA developed in the present invention can be used to change the genomic DNA sequence of a multicellular organism, including a eukaryotic one. To introduce the KbCas12d system in combination with the guide RNA/sgRNA into the cells of this organism (in all cells or in a part of the cells), various approaches known to those skilled in the art can be applied. For example, methods for delivering CRISPR-Cas systems to cells of organisms are disclosed in sources (Liu et al., 2017; Lino et al., 2018) and in sources disclosed within these sources.

For efficient expression of the KbCas12d nuclease in eukaryotic cells, it will be desirable to perform codon optimization for the amino acid sequence of the KbCas12d protein by methods known to those skilled in the art (eg, IDT codon optimization tool).

For the effective operation of the KbCas12d nuclease in eukaryotic cells, it is necessary to ensure the import of this protein into the nucleus of the eukaryotic cell. This can be done using the nuclear localization signal from the SV40 T-antigen (Lanford et al., Cell, 1986, 46: 575-582) coupled to the KbCas12d sequence with or without the spacer sequence described in Shen et al., 2013 . Thus, the complete amino acid sequence of a nuclease transported into the nucleus of a eukaryotic cell will be the following sequence: MAPKKKRKVGIHGVPAA-KbCas12d-KRPAATKKAGQAKKKK (SEQ ID NO: 15 - KbCas12d - SEQ ID NO: 16; hereinafter KbCas12d NLS). To deliver a protein with the above amino acid sequence, at least two approaches can be used.

Delivery as a gene is accomplished by creating a plasmid carrying the KbCas12d NLS gene under the regulation of a promoter (eg CMV promoter) and a sequence encoding guide RNAs under the regulation of the U6 promoter. As DNA targets, DNA sequences flanked by 5'-TA -3' are used, for example, the sequences of the human grin2b gene:

5'-AAATAGGATCTACATCAC-3'

Thus, the sgRNA expression cassette (SEQ ID NO: 17) is as follows:

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgaaagggtttccagtttttaactaaactttagccttccaccctttgaaagaaactccgaaagtatcggggataaaggcATCAATACCAAACTCTGG

The U6 promoter sequence is shown in bold, the sequence that forms the sgRNA structure is in lowercase letters, and the sequence necessary for recognition of the target DNA is in capital letters.

Plasmid DNA was purified and transfected into human HEK293 cells using the Lipofectamine 2000 reagent (Thermo Fisher Scientific). Cells are incubated for 72 hours, after which genomic DNA is isolated using genomic DNA purification columns (Thermo Fisher Scientific). The target DNA site is analyzed by sequencing on the Illumina platform to determine the number of DNA insertion-deletions that occur at the target site due to directed double-strand break and its subsequent repair.

For amplification of the target fragments, primers flanking the presumed site of the break are used.

After amplification, samples are prepared using the Ultra II DNA Library Prep Kit for Illumina (NEB) reagent protocol to prepare samples for high throughput sequencing. This is followed by sequencing on the Illumina 300cycles platform, direct reading. The sequencing results are analyzed by bioinformatic methods. The insertion or deletion of several nucleotides in the target DNA sequence is taken as a cut detection.

Delivery in the form of a ribonucleic complex is carried out by incubation of the recombinant form of KbCas12d NLS with guide RNAs in CutSmart Buffer (NEB). The recombinant protein is obtained from bacterial producer cells by purifying it by size separation affinity chromatography (NiNTA, Qiagen) (Superdex 200).

The protein is mixed with RNA in a ratio of 1:2 (KbCas9 NLS : sgRNA), incubated for 10 minutes at room temperature, then the mixture is transfected into cells.

Next, the DNA extracted from them is analyzed for insertions-deletions in the target DNA site (as described above).

The nuclease KbCas12d from Katanobacteria characterized in the present invention has a number of advantages over previously characterized Cas proteins.

To date, KbCas12d is the second Cas12d protein whose activity has been restored in vitro and the first Cas12d protein for which a hybrid sgRNA has been created. The use of sgRNA instead of a complex of crRNA and scout RNA simplifies the system and is an advantage over Cas12d15 from termite symbiont bacteria.

KbCas12d has a small protein size (1125 aa), which is also an advantage over other Cas nucleases. Effective cutting of DNA in a wide temperature range makes KbCas12d applicable both in fish and plant cells, and in cells of warm-blooded animals. Thus, KbCas12d in combination with the created hybrid RNA can become the basis of a new tool for genomic editing.

While the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific instances described in detail are for the purpose of illustrating the present invention only and should not be construed as limiting the scope of the invention in any way. It should be clear that it is possible to carry out various modifications without departing from the essence of the present invention.

Bibliography

Altschul S. F. et al., Basic local alignment search tool // J. Mol. Biol., 1990, Oct. 215(3): 403-410.

Brouns S. J. J. et al., Small CRISPR RNAs guide antiviral defense in prokaryotes // Science, 2008, 321 (5891): 960-964.

Harrington L. B. et al. A scoutRNA is required for some type V CRISPR-Cas systems // Mol. Cell, 2020, Aug, 79(3): 416-424.

Jansen R. et al., Identification of genes that are associated with DNA repeats in prokaryotes // Molecular microbiology, 2002, Mar, 43 (6): 1565-1575.

Lino C. A. et al., Delivering CRISPR: a review of the challenges and approaches // Drug Deliv., 2018, Nov, 25 (1): 1234-1257.

Liu C. et al., Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications // J Control Release. 2017, Nov. 28 (266): 17-26.

Mojica F. J. M. et al., Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements // Journal of molecular evolution. 2005, 60(2): 174-182.

Sambrook J. et al., Molecular Cloning: A Laboratory Manual, 1989, CSH Press, pp. 15.3-15.108.

Shen B., et al., Generation of gene-modified mice via Cas9/RNA-mediated gene targeting // Cell Res. 2013 May, 23(5): 720-3.

--->

<110> Federal State Budgetary Institution of Science Institute

Gene Biology of the Russian Academy of Sciences (Institute of Gene Biology Russian

Academy of Sciences

<120> Double strand DNA cutter with Cas12d protein from

Katanobacteria and hybrid RNA obtained by guide fusion

CRISPR RNA and scout RNA

<160> 17

<210> 1

<211> 1125

<212> PRT

<213> Katanobateria

<400> 1

Met Arg Lys Lys Leu Phe Lys Gly Tyr Ile Leu His Asn Lys Arg Leu 16

5 10 15

Val Tyr Thr Gly Lys Ala Ala Ile Arg Ser Ile Lys Tyr Pro Leu Val 32

20 25 30

Ala Pro Asn Lys Thr Ala Leu Asn Asn Leu Ser Glu Lys Ile Ile Tyr 48

35 30 45

Asp Tyr Glu His Leu Phe Gly Pro Leu Asn Val Ala Ser Tyr Ala Arg 64

50 55 60

Asn Ser Asn Arg Tyr Ser Leu Val Asp Phe Trp Ile Asp Ser Leu Arg 80

65 70 75 80

Ala Gly Val Ile Trp Gln Ser Lys Ser Thr Ser Leu Ile Asp Leu Ile 96

85 90 95

Ser Lys Leu Glu Gly Ser Lys Ser Pro Ser Glu Lys Ile Phe Glu Gln 112

100 105 110

Ile Asp Phe Glu Leu Lys Asn Lys Leu Asp Lys Glu Gln Phe Lys Asp 128

115 120 125

Ile Ile Leu Leu Asn Thr Gly Ile Arg Ser Ser Ser Asn Val Arg Ser 144

130 135 140

Leu Arg Gly Arg Phe Leu Lys Cys Phe Lys Glu Glu Phe Arg Asp Thr 160

145 150 155 160

Glu Glu Val Ile Ala Cys Val Asp Lys Trp Ser Lys Asp Leu Ile Val 176

165 170 175

Glu Gly Lys Ser Ile Leu Val Ser Lys Gln Phe Leu Tyr Trp Glu Glu 192

180 185 190

Glu Phe Gly Ile Lys Ile Phe Pro His Phe Lys Asp Asn His Asp Leu 208

195 200 205

Pro Lys Leu Thr Phe Phe Val Glu Pro Ser Leu Glu Phe Ser Pro His 224

210 215 220

Leu Pro Leu Ala Asn Cys Leu Glu Arg Leu Lys Lys Phe Asp Ile Ser 240

225 230 235 240

Arg Glu Ser Leu Leu Gly Leu Asp Asn Asn Phe Ser Ala Phe Ser Asn 256

245 250 255

Tyr Phe Asn Glu Leu Phe Asn Leu Leu Ser Arg Gly Glu Ile Lys Lys 272

260 265 270

Ile Val Thr Ala Val Leu Ala Val Ser Lys Ser Trp Glu Asn Glu Pro 288

275 280 285

Glu Leu Glu Lys Arg Leu His Phe Leu Ser Glu Lys Ala Lys Leu Leu 304

290 295 300

Gly Tyr Pro Lys Leu Thr Ser Ser Trp Ala Asp Tyr Arg Met Ile Ile 320

305 310 315 320

Gly Gly Lys Ile Lys Ser Trp His Ser Asn Tyr Thr Glu Gln Leu Ile 336

325 330 335

Lys Val Arg Glu Asp Leu Lys Lys His Gln Ile Ala Leu Asp Lys Leu 352

340 345 350

Gln Glu Asp Leu Lys Lys Val Val Asp Ser Ser Leu Arg Glu Gln Ile 368

355 360 365

Glu Ala Gln Arg Glu Ala Leu Leu Pro Leu Leu Asp Thr Met Leu Lys 384

370 375 380

Glu Lys Asp Phe Ser Asp Asp Leu Glu Leu Tyr Arg Phe Ile Leu Ser 400

385 390 395 400

Asp Phe Lys Ser Leu Leu Asn Gly Ser Tyr Gln Arg Tyr Ile Gln Thr 416

405 410 415

Glu Glu Glu Arg Lys Glu Asp Arg Asp Val Thr Lys Lys Tyr Lys Asp 432

420 425 430

Leu Tyr Ser Asn Leu Arg Asn Ile Pro Arg Phe Phe Gly Glu Ser Lys 448

435 440 445

Lys Glu Gln Phe Asn Lys Phe Ile Asn Lys Ser Leu Pro Thr Ile Asp 464

450 455 460

Val Gly Leu Lys Ile Leu Glu Asp Ile Arg Asn Ala Leu Glu Thr Val 480

465 470 475 480

Ser Val Arg Lys Pro Pro Ser Ile Thr Glu Glu Tyr Val Thr Lys Gln 496

485 490 495

Leu Glu Lys Leu Ser Arg Lys Tyr Lys Ile Asn Ala Phe Asn Ser Asn 512

500 505 510

Arg Phe Lys Gln Ile Thr Glu Gln Val Leu Arg Lys Tyr Asn Asn Gly 528

515 520 525

Glu Leu Pro Lys Ile Ser Glu Val Phe Tyr Arg Tyr Pro Arg Glu Ser 544

530 535 540

His Val Ala Ile Arg Ile Leu Pro Val Lys Ile Ser Asn Pro Arg Lys 560

545 550 555 560

Asp Ile Ser Tyr Leu Leu Asp Lys Tyr Gln Ile Ser Pro Asp Trp Lys 576

565 570 575

Asn Ser Asn Pro Gly Glu Val Val Asp Leu Ile Glu Ile Tyr Lys Leu 592

580 585 590

Thr Leu Gly Trp Leu Leu Ser Cys Asn Lys Asp Phe Ser Met Asp Phe 608

595 600 605

Ser Ser Tyr Asp Leu Lys Leu Phe Pro Glu Ala Ala Ser Leu Ile Lys 624

610 615 620

Asn Phe Gly Ser Cys Leu Ser Gly Tyr Tyr Leu Ser Lys Met Ile Phe 640

625 630 635 640

Asn Cys Ile Thr Ser Glu Ile Lys Gly Met Ile Thr Leu Tyr Thr Arg 656

645 650 655

Asp Lys Phe Val Val Arg Tyr Val Thr Gln Met Ile Gly Ser Asn Gln 672

660 665 670

Lys Phe Pro Leu Leu Cys Leu Val Gly Glu Lys Gln Thr Lys Asn Phe 688

675 680 685

Ser Arg Asn Trp Gly Val Leu Ile Glu Glu Lys Gly Asp Leu Gly Glu 704

690 695 700

Glu Lys Asn Gln Glu Lys Cys Leu Ile Phe Lys Asp Lys Thr Asp Phe 720

705 710 715 720

Ala Lys Ala Lys Glu Val Glu Ile Phe Lys Asn Asn Ile Trp Arg Ile 736

725 730 735

Arg Thr Ser Lys Tyr Gln Ile Gln Phe Leu Asn Arg Leu Phe Lys Lys 752

740 745 750

Thr Lys Glu Trp Asp Leu Met Asn Leu Val Leu Ser Glu Pro Ser Leu 768

755 760 765

Val Leu Glu Glu Glu Glu Trp Gly Val Ser Trp Asp Lys Asp Lys Leu Leu 784

770 775 780

Pro Leu Leu Lys Lys Glu Lys Ser Cys Glu Glu Arg Leu Tyr Tyr Ser 800

785 790 795 800

Leu Pro Leu Asn Leu Val Pro Ala Thr Asp Tyr Lys Glu Gln Ser Ala 816

805 810 815

Glu Ile Glu Gln Arg Asn Thr Tyr Leu Gly Leu Asp Val Gly Glu Phe 832

820 825 830

Gly Val Ala Tyr Ala Val Val Arg Ile Val Arg Asp Arg Ile Glu Leu 848

835 840 845

Leu Ser Trp Gly Phe Leu Lys Asp Pro Ala Leu Arg Lys Ile Arg Glu 864

850 855 860

Arg Val Gln Asp Met Lys Lys Lys Gln Val Met Ala Val Phe Ser Ser 880

865 870 875 880

Ser Ser Thr Ala Val Ala Arg Val Arg Glu Met Ala Ile His Ser Leu 896

885 890 895

Arg Asn Gln Ile His Ser Ile Ala Leu Ala Tyr Lys Ala Lys Ile Ile 912

900 905 910

Tyr Glu Ile Ser Ile Ser Asn Phe Glu Thr Gly Gly Asn Arg Met Ala 928

915 920 925

Lys Ile Tyr Arg Ser Ile Lys Val Ser Asp Val Tyr Arg Glu Ser Gly 944

930 935 940

Ala Asp Thr Leu Val Ser Glu Met Ile Trp Gly Lys Lys Asn Lys Gln 960

945 950 955 960

Met Gly Asn His Ile Ser Ser Tyr Ala Thr Ser Tyr Thr Cys Cys Asn 976

965 970 975

Cys Ala Arg Thr Pro Phe Glu Leu Val Ile Asp Asn Asp Lys Glu Tyr 992

980 985 990

Glu Lys Gly Gly Asp Glu Phe Ile Phe Asn Val Gly Asp Glu Lys Lys 1008

995 1000 1005

Val Arg Gly Phe Leu Gln Lys Ser Leu Leu Gly Lys Thr Ile Lys Gly 1024

1010 1015 1020

Lys Glu Val Leu Lys Ser Ile Lys Glu Tyr Ala Arg Pro Pro Ile Arg 1040

1025 1030 1035 1040

Glu Val Leu Leu Glu Gly Glu Asp Val Glu Gln Leu Leu Lys Arg Arg 1056

1045 1050 1055

Gly Asn Ser Tyr Ile Tyr Arg Cys Pro Phe Cys Gly Tyr Lys Thr Asp 1072

1060 1065 1070

Ala Asp Ile Gln Ala Ala Leu Asn Ile Ala Cys Arg Gly Tyr Ile Ser 1088

1075 1080 1085

Asp Asn Ala Lys Asp Ala Val Lys Glu Gly Glu Arg Lys Leu Asp Tyr 1104

1090 1095 1100

Ile Leu Glu Val Arg Lys Leu Trp Glu Lys Asn Gly Ala Val Leu Arg 1120

1105 1110 1115 1120

Ser Ala Lys Phe Leu 1125

1125

<210> 2

<211> 59

<212> RNA

<213> artificial sequence

<220>

<223> KbCas12d scout RNA

<400> 2

aaguaucaaa auaaaaaggg uuuccaguuu uuaacuaaac uuuagccuuc cacccuuuc 59

<210> 3

<211> 44

<212> RNA

<213> artificial sequence

<220>

<223> KbCas12d crRNA, where n is any nucleotide

<400> 3

acuccgaaag uaucggggau aaaggcnnnn nnnnnnnnnn nnnn 44

<210> 4

<211> 916

<212> DNA

<213> artificial sequence

<220>

<223> linear DNA fragment

<400> 4

ccctgcaaac acaaagaaag agcatgttaa aataggatct acatcacgta acctgtctta 60

gaagaggcta gatactgcaa ttcaaggacc ttatctcctt tcattgagca ccaaacccaa 120

ctccatctac cagcctactc tcttatctct ggtatttgct ctgcagaatg agagaaaatg 180

aaactttcaa aagcctcaga aatccttgaa caaggcaata aaaggtgcta ttgctatagt 240

cattggcagc tacaggcaga gacaaaggag gaaaagaggt tgtgagtggt ccaggtagcc 300

atgcgagtat gcatacacaa atctcctggc cctcctgtta cagcccaccc ttgtactgtt 360

cttgggctga aggaaagcaa ggccagacaa agtgagcaga aaaacgtgct cagcagaggt 420

gagcaacaga acatgggctg gataaactgg atgtgggggg ctataagtac acaagccctg 480

cattcttgct gccttcacct tatgtttgcc tcaatgagga caacagccag aaaattcttt 540

agtaaccttg ttagtatctg gctcttaata ttaaactaca agaacaactg atacatgact 600

agtagtttta aaacattgcc tcaattgatc cttacaatga cccagtaggg aataaaataa 660

gaaaaacatt attatcacca tttttataaa tgttgacgcc aaggctcaga gaagctaagt 720

gttctaagac catgaaccaa tcaattaagt gaaacaaacc tgaacccaga tcttctgact 780

tttgtattcc aatatgtgtt ctattacact acgtggaact gcctctcata taaccaatta 840

ttataaggaa cacatattac tccaatctat ttatacacca aaatacagaa acttaaacaa 900

ataccattag cagctg 916

<210> 5

<211> 43

<212> RNA

<213> artificial sequence

<220>

<223> crRNA

<400> 5

cuccgaaagu aucggggaua aaggcgucau uggcagcuac agg 43

<210> 6

<211> 44

<212> RNA

<213> artificial sequence

<220>

<223> scout RNA

<400> 6

aaaggguuuc caguuuuuaa cuaaacuuua gccuuccacc cuuu 44

<210> 7

<211> 62

<212> RNA

<213> artificial sequence

<220>

<223> scout RNA

<400> 7

60

ug 62

<210> 8

<211> 76

<212> RNA

<213> artificial sequence

<220>

<223> scout RNA

<400> 8

uuuccaguuu uuaacuaaac uuuagccuuc cacccuuucc 60

ugauuuuguu gauaau 76

<210> 9

<211> 59

<212> RNA

<213> artificial sequence

<220>

<223> scout RNA

<400> 9

aaguaucaaa auaaaaaggg uuuccaguuu uuaacuaaac uuuagccuuc cacccuuuc 59

<210> 10

<211> 62

<212> RNA

<213> artificial sequence

<220>

<223> scout RNA

<400> 10

auuaucaaca aaaucaggaa aggguggaag gcuaaaguuu aguuaaaaac uggaaacccu 60

uu 62

<210> 11

<211> 91

<212> RNA

<213> artificial sequence

<220>

<223> sgRNA

<400> 11

aaaggguuuc caguuuuuaa cuaaacuuua gccuuccacc cuuugaaacu ccgaaaguau 60

cggggauaaa ggcaucaua ccaaacucug g 91

<210> 12

<211> 95

<212> RNA

<213> artificial sequence

<220>

<223> sgRNA, regions of the spacer sequence (from 78 to 95 bp

sequence aucauaccaaacucugg) and a linker (bp 45 to 52).

sequence gaaagaaa) can be variable

<400> 12

aaaggguuuc caguuuuuaa cuaaacuuua gccuuccacc cuuugaaaga aacuccgaaa 60

guaucgggga uaaaggcauc aauaccaaac ucugg 95

<210> 13

<211> 99

<212> RNA

<213> artificial sequence

<220>

<223> sgRNA

<400> 13

aaaggguuuc caguuuuuaa cuaaacuuua gccuuccacc cuuugaaaga aagaaacucc 60

gaaaguaucg gggauaaagg caucaauacc aaacucugg 99

<210> 14

<211> 1612

<212> DNA

<213> H. sapiens

<223> grin2b gene fragment

<400> 14

gagagagatg gccaaggctt atattctata gagcattatg tccttagttt gatgcataga 60

ataagattta gggtcatatg tggaagtaaa aaggaaggag ttctttgtag gtaaaaggtg 120

gcaaattata tgaaaatacg gtatcagtca ttttagggaa gtcacgacta taggatggca 180

tcagaccttt tattgccttg ttcagaaaaa aaaaggaaca tttttcaaat gtggctctaa 240

cattacttca gctgctaatg gtatttgttt aagtttctgt attttggtgt ataaatagat 300

tggagtaata tgtgttcctt ataataattg gttatatgag aggcagttcc acgtagtgta 360

420

ttgattggtt catggtctta gaacacttag cttctctgag ccttggcgtc aacatttata 480

540

ttgaggcaat gttttaaaac tactagtcat gtatcagttg ttcttgtagt ttaatattaa 600

gagccagata ctaacaaggt tactaaagaa ttttctggct gttgtcctca ttgaggcaaa 660

cataaggtga aggcagcaag aatgcagggc ttgtgtactt atagcccccc acatccagtt 720

tatccagccc atgttctgtt gctcacctct gctgagcacg tttttctgct cactttgtct 780

ggccttgctt tccttcagcc caagaacagt acaagggtgg gctgtaacag gagggccagg 840

agatttgtgt atgcatactc gcatggctac ctggaccact cacaacctct tttcctcctt 900

tgtctctgcc tgtagctgcc aatgactata gcaatagcac cttttattgc cttgttcaag 960

gatttctgag gcttttgaaa gtttcatttt ctctcattct gcagagcaaa taccagagat 1020

aagagagtag gctggtagat ggagttggggt ttggtgctca atgaaaggag ataaggtcct 1080

tgaattgcag tatctagcct cttctaagac aggttacgtg atgtagatcc tattttaaca 1140

tgctctttct ttgtgtttgc agggagtcga cgagttgaag atgaagccca gagcggagtg 1200

ctgttctccc aagttctggt tggtgttggc cgtcctggcc gtgtcaggca gcagagctcg 1260

ttctcagaag agccccccca gcattggcat tgctgtcatc ctcgtgggca cttccgacga 1320

ggtggccatc aaggatgccc acgagaaaga tgatttccac catctctccg tggtaccccg 1380

ggtggaactg gtagccatga atgagaccga cccaaagagc atcatcaccc gcatctgtga 1440

tctcatgtct gaccggaaga tccagggggt ggtgtttgct gatgacacag accaggaagc 1500

catcgcccag atcctcgatt tcatttcagc acagactctc acccccatcc tgggcatcca 1560

cgggggctcc tctatgataa tggcagataa ggtaaaaagg ggctgcaggg ag 1612

<210> 15

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> nuclease part

<400> 15

Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 16

1 5 10 15

Ala 17

<210> 16

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> nuclease part

<400> 16

Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 16

<210> 17

<211>

<212> DNA

<213> artificial sequence

<220>

<223> sgRNA expression cassette

<400> 17

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg aaagggtttc cagtttttaa ctaaacttta gccttccacc ctttgaaaga 300

aactccgaaa gtatcgggga taaaggcaaa taggatctac atcac 345

<---

Claims (9)

1. Hybrid RNA (sgRNA) used as a guide RNA in the Cas12d system for editing genomic DNA, which is a nucleic acid molecule obtained by fusing the nucleotide sequences of scout RNA (scout RNA) and crRNA (crRNA), with the general formula A-B- C-D, where A is the KbCas12d (CRISPR-Cas12d system from Katanobacteria) scout RNA sequence, B is the linker sequence; C, direct repeat sequence of DR KbCas12d crRNA; D, sequence complementary to the target DNA (spacer segment); while the sequence of KbCas12d scout RNA is characterized by SEQ ID NO: 6, and the sequence of the linker and spacer segment can be any. 2. The fusion RNA according to claim 1, wherein the KbCas12d crRNA direct repeat DR sequence is a direct repeat sequence of SEQ ID NO: 3. 3. Hybrid RNA according to claim 1, which is the sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, but not limited to them. 4. DNA cassette for the expression of the hybrid RNA according to claim 1, consisting of the nucleotide sequence of the U6 promoter, the nucleotide sequence encoding the hybrid RNA according to claim 1, and the nucleotide sequence necessary for recognition of the target DNA and the sequence 5 flanked from the 5' end of the PAM '-TA -3'. 5. A method for altering the genomic DNA sequence of a unicellular or multicellular organism, comprising introducing into at least one cell of this organism an effective amount of: a) a protein having a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding a protein gene having a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1; b) hybrid RNA according to claim 1, in this case, the interaction of the specified protein with the hybrid RNA and the 5'-TA-3' nucleotide sequence leads to the formation of a double-strand break in the genomic DNA sequence immediately adjacent to the 5'-TA-3' sequence. 6. The method of claim 8, further comprising introducing the exogenous DNA sequence simultaneously with the fusion RNA.

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