CN116694603A - Novel Cas protein, crispr-Cas system and use thereof in the field of gene editing - Google Patents

Abstract

The invention relates to the field of gene editing, in particular to a novel Cas protein, a Crispr-Cas system and application thereof in the field of gene editing. The novel Cas protein is selected from at least one of the following: SEQ ID NO. 1-SEQ ID NO. 4; the sequence similarity is 85% or more, preferably 90% or more, compared with any one of SEQ ID NO 1 to SEQ ID NO 4. The novel Cas protein provided by the invention can be used for a Crispr-Cas system, and can be used for editing genes. It can edit more target sites and is easier to deliver into cells for editing without causing off-target.

Description

Novel Cas proteins, Crispr-Cas systems and their applications in gene editing

Technical Field

The present invention relates to the field of gene editing, and in particular to a novel Cas protein, a Crispr-Cas system and uses thereof in the field of gene editing.

Background Art

CRISPR (Clustered regularly interspaced short palindromic repeats), known as regularly clustered short palindromic repeats, is actually a gene editor and a natural immunity method in most bacteria and archaea. Through the analysis of the flanking sequences of the CRISPR cluster, it was found that there is a polymorphic family gene near it, and it works together with the CRISPR region, so it is named CRISPR-associated gene (CRISPR-associated), abbreviated as Cas. Most CRISPR-Cas systems contain cas1 protein, and cas1 is a more conservative protein in the Cas family. According to the structure of the effector module, there are two main types of CRISPR-Cas systems discovered so far: Class 1 contains multiple Cas proteins and multiple effector proteins (effectors) work together, mainly including Type I, Type III and Type IV; Class 2 contains only a huge effector protein, including Type II, Type V and Type VI. At present, Class 2 includes Cas9 system (Type II) and Cpf1 (Type V) system, and is widely used in gene editing applications.

However, the Crispr-Cas system still has many shortcomings, such as the possibility of gene off-target, and its scope of application is limited, and further improvement is needed.

Summary of the invention

The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one object of the present invention is to propose a novel Cas protein, Crispr-Cas system and its use in the field of gene editing.

The CRISPR/Cas system is a commonly used gene editing system that can be successfully applied to the precise editing of animal and plant genomes. The system is mediated by RNA to target and recognize specific sites of DNA double strands and cut them through nucleases. The most widely used ones are Cas9 nucleases and Cpf1 nucleases. Cas9 nucleases and Cpf1 nucleases target and recognize specific sites of DNA double strands and cut them through RNA, causing DNA double strand breaks. The cells then repair them through NHEJ (nonhomologous end joining) or HR (homologous recombination) pathways to achieve site-specific modification of the target gene. A widely used Cas9 nuclease in commercial applications is SpCas9 nuclease, which recognizes the PAM sequence NGG, located at the 3' end of the target sequence, and cuts at 3bp from the PAM sequence to form a blunt end. LbCpf1 is a widely used Cpf1 nuclease in commercial applications. Its recognition PAM site is the TTTN sequence located at the 5' end of the target sequence, and cuts at the distal end to form a sticky end.

During the research, it was found that both SpCas9 and LbCpf1 have relatively strict PAM sequences, which restrict the design of targeting sites. Moreover, SpCas9 protein and LbCpf1 protein are composed of 1368 and 1228 amino acids respectively, which are too large to be packaged and delivered by AAV virus, which limits their application in animal cells to a certain extent. Moreover, the targeting sequence of SpCas9 is 20bp, and similar sequences are likely to appear in the whole genome, resulting in off-target.

It is crucial to find new available Cas proteins that are smaller in length, so that they can be packaged and delivered more easily, further expanding their applications in animal cells. It is also crucial to make the Crispr-Cas system less likely to cause off-target effects.

To this end, through research we have found a variety of new Cas proteins with shorter protein lengths. When used in the Crispr-Cas system, they can be more easily delivered to cells for editing. And it is less likely to be off-target. Taking the BES1 protein obtained from the human intestinal bacteria Veillonella sp AF13-2 (hereinafter referred to as: AF13-2) as an example, it is used in the Crispr-Cas system, and the specificity of the PAM sequence recognized is lower than that of the commercial SpCas9 and LbCpf1. The Cas protein has more potential target sites that can be edited. Moreover, the BES1 protein consists of only 1064 amino acids, which is easier to be delivered to cells for editing. The targeting sequence of SpCas9 is 20bp, and the targeting sequence of our BES1 is 23bp, which is potentially less likely to cause off-target than SpCas9.

Specifically, the present invention provides the following technical solutions:

According to the first aspect of the present invention, the present invention provides a Cas protein, selected from at least one of the following: SEQ ID NO: 1 to SEQ ID NO: 4; compared with any sequence in SEQ ID NO: 1 to SEQ ID NO: 4, the sequence similarity is more than 85%, preferably more than 90%. After biological information technology screening, a new type of Cas protein SEQ ID NO: 1 to SEQ ID NO: 4 was obtained, and it was verified by molecular biology technology. Any of these Cas proteins is easily delivered to cells for gene editing. Moreover, the PAM sequence it recognizes is specific, so more target sites can be edited, and the length of the targeting sequence is also suitable, which is less likely to cause off-target. Compared with any protein in SEQ ID NO:1 to SEQ ID NO:4, the sequence similarity is above 85%, for example, above 86%, above 87%, above 88%, above 89%, preferably above 90%, for example, above 91%, above 92%, above 93%, above 94%, which has the same or similar activity and function as the Cas protein shown in SEQ ID NO:1 to SEQ ID NO:4, and is also easily delivered to cells for gene editing, and more target sites can be edited, the length of the targeted sequence is more suitable, and it is less likely to cause off-target.

According to an embodiment of the present invention, the Cas protein described above may further include the following technical features:

In some embodiments of the present invention, compared with any sequence in SEQ ID NO: 1 to SEQ ID NO: 4, the sequence similarity is more than 95%, preferably more than 96%, more preferably more than 97%, more preferably more than 98%, and most preferably more than 99%. Compared with any protein in SEQ ID NO: 1 to SEQ ID NO: 4, the protein with a sequence similarity of more than 95%, preferably more than 96%, more than 97%, more than 98%, more than 99%, and more than 99.5% has the same or similar activity as the above-mentioned Cas protein, is easily delivered to cells for gene editing, and can edit more target sites, and the length of the targeted sequence is more suitable, which is not easy to cause off-target.

In some embodiments of the present invention, the Cas protein is a Cas protein having nuclease activity after substitution, deletion or addition of one or more amino acids compared to any sequence in SEQ ID NO: 1 to SEQ ID NO: 4. These proteins have the same or similar nuclease activity as the proteins shown in SEQ ID NO: 1 to SEQ ID NO: 4, are easily delivered to cells for gene editing, have multiple target sites, and are not easy to be off-target.

In some embodiments of the present invention, the Cas protein is a Cas protein with nuclease activity after substitution, deletion or addition of up to 8 amino acids compared to any sequence in SEQ ID NO: 1 to SEQ ID NO: 4. These proteins have the same or similar nuclease activity as the proteins shown in SEQ ID NO: 1 to SEQ ID NO: 4, are easily delivered to cells for gene editing, have multiple target sites, and are not easy to be off-target.

In some embodiments of the present invention, the Cas protein is a Cas protein with nuclease activity after substitution, deletion or addition of up to 6 amino acids compared to any sequence in SEQ ID NO: 1 to SEQ ID NO: 4. These proteins have the same or similar nuclease activity as the proteins shown in SEQ ID NO: 1 to SEQ ID NO: 4, are easily delivered to cells for gene editing, have multiple target sites, and are not easy to be off-target.

In some embodiments of the present invention, the Cas protein is a Cas protein with nuclease activity after substitution, deletion or addition of up to 5 amino acids compared to any sequence in SEQ ID NO: 1 to SEQ ID NO: 4. These proteins have the same or similar nuclease activity as the proteins shown in SEQ ID NO: 1 to SEQ ID NO: 4, are easily delivered to cells for gene editing, have multiple target sites, and are not easy to be off-target.

In some embodiments of the present invention, the Cas protein is a Cas protein with nuclease activity after substitution, deletion or addition of up to 4 amino acids compared to any sequence in SEQ ID NO: 1 to SEQ ID NO: 4. These proteins have the same or similar nuclease activity as the proteins shown in SEQ ID NO: 1 to SEQ ID NO: 4, are easily delivered to cells for gene editing, have multiple target sites, and are not easy to be off-target.

In some embodiments of the present invention, the Cas protein is a Cas protein with nuclease activity after substitution, deletion or addition of up to 3 amino acids compared to any sequence in SEQ ID NO: 1 to SEQ ID NO: 4. These proteins have the same or similar nuclease activity as the proteins shown in SEQ ID NO: 1 to SEQ ID NO: 4, are easily delivered to cells for gene editing, have multiple target sites, and are not easy to be off-target.

In some embodiments of the present invention, the Cas protein is a Cas protein with nuclease activity after substitution, deletion or addition of up to 2 amino acids compared to any sequence in SEQ ID NO: 1 to SEQ ID NO: 4. These proteins have the same or similar nuclease activity as the proteins shown in SEQ ID NO: 1 to SEQ ID NO: 4, are easily delivered to cells for gene editing, have multiple target sites, and are not easy to be off-target.

In some embodiments of the present invention, the Cas protein is a Cas protein with nuclease activity after substitution, deletion or addition of one amino acid compared with any sequence in SEQ ID NO: 1 to SEQ ID NO: 4. These proteins have the same or similar nuclease activity as the proteins shown in SEQ ID NO: 1 to SEQ ID NO: 4, are easily delivered to cells for gene editing, have multiple target sites, and are not easy to be off-target.

In some embodiments of the present invention, the Cas protein is shown in SEQ ID NO: 1. The Cas protein consists of 1064 amino acids, which has a relatively small number of amino acids and is easier to be delivered to cells for editing. The PAM sequence it recognizes is NNNV (where V represents the base A/G/C), so more target sites can be edited, and its targeting sequence is 23bp, which is not easy to cause off-target phenomena. The Cas protein has the activity of cutting double-stranded DNA in vitro, and no editing activity in human cells has been detected.

In some embodiments of the present invention, the Cas protein is shown in SEQ ID NO: 2. The Cas protein consists of 1368 amino acids, which has a relatively small number of amino acids and is easier to be delivered into cells for editing, and the PAM sequence it recognizes is NMNTA. The Cas protein has in vitro DNA double-strand cleavage activity, and no editing activity in human cells has been detected.

In some embodiments of the present invention, the Cas protein is shown in SEQ ID NO: 3. The Cas protein consists of 1245 amino acids, which has a relatively small number of amino acids and is easier to be delivered into cells for editing, and the PAM sequence it recognizes is TTTN. The Cas protein has in vitro DNA double-strand cleavage activity and editing activity in human cells.

In some embodiments of the present invention, the Cas protein is shown in SEQ ID NO: 4. The Cas protein consists of 1306 amino acids, which has a relatively small number of amino acids and is easier to be delivered into cells for editing. Moreover, the PAM sequence it recognizes is YYN, which greatly alleviates the limitation that LbCpf1 only recognizes TTTN. The Cas protein has in vitro DNA double-strand cleavage activity and editing activity in human cells.

According to the second aspect of the present invention, the present invention provides a nucleic acid sequence, which is selected from at least one of the following: a nucleic acid sequence encoding the Cas protein described in any embodiment of the first aspect of the present invention; a nucleic acid sequence that is reverse complementary to the nucleic acid sequence encoding the Cas protein described in any embodiment of the first aspect of the present invention.

In some embodiments of the present invention, the nucleic acid sequence is DNA or RNA.

According to the third aspect of the present invention, the present invention provides an expression vector, the expression vector comprising the nucleic acid sequence described in the second aspect of the present invention. The above nucleic acid sequence is constructed with a vector to obtain an expression vector, which can express the corresponding Cas protein in the target cell, thereby performing corresponding gene editing in the target cell. Commonly used vectors can be plasmids, lentiviruses, etc., for example, pET 28a vectors, pMD19 vectors, etc.

According to a fourth aspect of the present invention, the present invention provides a recombinant cell, which contains the expression vector described in the third aspect of the present invention. The expression vector is introduced into the cell to form a recombinant cell, and the corresponding Cas protein is expressed by the expression vector, so that gene editing of the recombinant cell can be achieved. These recombinant cells can be eukaryotic cells, such as plant cells and animal cells. In particular, compared with the commonly used SpCas9 protein and LbCpf1 protein, the Cas protein provided herein has fewer amino acids and is easier to be delivered to cells for editing. When used in animal cells, it is more convenient to be packaged and delivered by viral vectors, which expands its application in the field of animal cells.

According to a fifth aspect of the present invention, the present invention provides a Crispr-Cas system, comprising the Cas protein described in the first aspect of the present invention. The Cas protein provided by the present invention can be used in the Crispr-Cas system, applied to the field of gene editing, expanding the editable range, and not easy to off-target, thereby improving the accuracy of editing. The system can be used in many fields such as basic biological sciences, medicine, and agriculture.

According to an embodiment of the present invention, the Crispr-Cas system described above may further include the following technical features:

In some embodiments of the present invention, the Crispr-Cas system further includes at least one of the following: crRNA, tracrRNA, or a chimeric RNA formed by crRNA and tracrRNA. These RNAs can help the Crispr-cas system to perform the gene editing function. In addition, the Crispr-cas system can further include a Crispr_repeat sequence as needed, wherein the Crispr_repeat sequence corresponding to each Cas protein is shown in Appendix I and Appendix II.

In some embodiments of the present invention, the crRNA and tracrRNA are shown in Appendix I and Appendix II. The crRNA and tracrRNA sequences used by the Cas protein in gene editing are listed in Appendix I and Appendix II. These sequences can help the Cas protein accurately locate the target sequence and achieve accurate gene editing.

According to the sixth aspect of the present invention, the present invention provides use of the Cas protein, nucleic acid sequence, expression vector, recombinant cell or Crispr-Cas system described in the first aspect of the present invention in the field of gene editing, wherein the Cas protein is the Cas protein described in the first aspect of the present invention, the nucleic acid sequence is the nucleic acid sequence described in the second aspect of the present invention, the expression vector is the expression vector described in the third aspect of the present invention, the recombinant cell is the recombinant cell described in the fourth aspect of the present invention, and the Crispr-Cas system is the Crispr-Cas system described in the fifth aspect of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a PAM preference diagram of BES1 provided according to an embodiment of the present invention.

FIG. 2 is a diagram showing the results of BES1 purification according to an embodiment of the present invention.

FIG3 is a base sequence and structure diagram of crRNA+tracrRNA-L, sgRNA-1, sgRNA-2 and sgRNA-3 of BES1 provided according to an embodiment of the present invention.

4 is a PAM preference diagram of BES1 detected by a chip using crRNA+tracrRNA-L, sgRNA-1, and sgRNA-3, respectively, according to an embodiment of the present invention.

FIG. 5 is a spacer sequence diagram provided according to an embodiment of the present invention.

FIG. 6 is a sequence of a PAM library constructed according to an embodiment of the present invention.

FIG. 7 is a schematic diagram of a cleavage substrate sequence provided according to an embodiment of the present invention.

Figure 8 is a band diagram of in vitro cleavage products of BES1 and crRNA+tracrRNA-L, sgRNA-1, sgRNA-2 and sgRNA-3 at 20°C, 25°C and 37°C according to an embodiment of the present invention.

FIG. 9 is a schematic diagram of a process for obtaining a novel Cas protein according to an embodiment of the present invention.

FIG. 10 is a PAM preference diagram of chip detection BES2, BES4 and BES6 systems according to an embodiment of the present invention.

FIG. 11 is a diagram of an in vitro cutting experiment of BES2, BES4 and BES6 systems according to an embodiment of the present invention.

FIG12 is an electrophoresis diagram of human cell editing activity detection in the BES6 system according to an embodiment of the present invention.

FIG13 is an electrophoresis diagram of human cell editing activity detection by the BES4 system according to an embodiment of the present invention.

DETAILED DESCRIPTION

Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limitations of the present invention. At the same time, in order to enable those skilled in the art to better understand the present invention, certain terms or expressions appearing in this document are explained, and these explanations and descriptions are only used to facilitate the understanding of the present invention, and should not be regarded as limitations on the scope of protection of the present invention.

As used herein, the term "Crispr", "crispr" or "CRISPR" refers to the acronym for Clustered regularly interspaced short palindromic repeats. The terms, whether in uppercase or lowercase or with the first letter capitalized, are commonly used expressions in the art. Accordingly, there are different expressions in the Crispr-Cas system due to the capitalization of letters. In addition, when representing bases, unless otherwise specified, the bases represented by the letters N and V have the usual meanings in the art, that is, N represents random or arbitrary bases A, T, C or G, and V represents random or arbitrary bases A, C or G.

The Cas9 enzyme cuts at the target DNA target site, and the target site is usually determined in the following way: an RNA molecule called Crispr RNA (crRNA) uses part of its sequence to combine with an RNA molecule called tracrRNA through base pairing to form a chimeric RNA (tracrRNA/crRNA), and then uses another part of the crRNA sequence to base pair with the target DNA site. As a result, the chimeric RNA guides the Cas protein to bind to this target site for cutting. This chimeric RNA is also called guide RNA. Unlike the Crispr-Cas9 system, the Cpf1 enzyme can process the crRNA precursor alone, and then use the processed crRNA to specifically target and cut DNA, without the need for ribonucleases and tracrRNA from host cells.

The targeting specificity of Crispr is determined by two parts: one is the base pairing between the RNA chimera and the target DNA, and the other relies on the Cas protein and a short DNA sequence at the 3’ end of the target DNA, called PAM (protospacer adjacent motif).

If the PAM sequence is strict (for example, it may be a few specific bases), the Cas protein can edit fewer target sites, thus limiting the application of the Crispr-Cas system. Both SpCas9 and LbCpf1 have relatively strict PAM sequences, which restricts the design of the target site. For example, the PAM sequence recognized by the SpCas9 nuclease is NGG, which is located at the 3' end of the target sequence and cuts at 3bp from the PAM sequence to form a flat end. Since its PAM sequence is only NGG, the application of this editing system is limited.

We have found a variety of new Cas9 systems and Cpf1 systems with gene editing potential in human intestinal flora using bioinformatics and molecular experimental techniques, as shown in Appendix I and Appendix II. Among them, the Cpf1 enzyme in the Cpf system, also known as the Cas12a protein, performs gene editing in a different way from the Cas9 protein. The Cpf1 enzyme is smaller than the SpCas9 protein and is easier to deliver to cells and tissues. Moreover, its application in the Crsipr-Cpf1 system only requires one crRNA, and simultaneous editing of multiple sites can be achieved. The Cas protein provided in this application includes both Cas9 protein and Cpf1 protein. That is, the present invention provides a Cas protein, which is at least one of SEQ ID NO:1 to SEQ ID NO:4. These Cas proteins have nuclease activity and can be used to cut target nucleic acids, so as to be applied to the Crispr-cas system to achieve effective gene editing, and there are more target sites that can be used for editing, and the application range is wider.

The new Cas9 and Cpf1 systems provided have lower PAM specificity, thus expanding the application of gene editing systems. Taking the BES1 protein we obtained on human intestinal bacteria Veillonella sp AF13-2 (hereinafter referred to as: AF13-2) as an example, it has lower PAM specificity and smaller protein than the existing commercial SpCas9 and LbCpf1. The number of amino acids in the BES1 protein is relatively small, and it is easier to be delivered to cells to perform gene editing functions. Moreover, the PAM sequence preference of BES1 is shown in Figure 1, where the abscissa in Figure 1 represents the 7 sites adjacent to the 3' end of the target sequence, and the ordinate represents the proportion of each base in all positive sequences cut. In Figure 1, at the first site adjacent to the 3' end of the target sequence, the probability of base A, base C, base T or base G is very large, and the site can be represented as N, and the results of each site are observed in turn. As can be seen from Figure 1, only when the fourth position is T, the probability of being cut is extremely low (less than 0.05), so the PAM sequence of BES1 is NNNV (where V represents the base A, G or C).

The new Cas9 system table includes the strain name where the Cas protein is located, Genome ID (NCBI database), TaxID (NCBI database), species, genus, phylum, crRNA, tracrRNA, crispr repeat sequence, effector protein length, effector amino acid sequence, etc. See Appendix I for details.

The new Cpf1 system table includes the strain name where the Cas protein is located, Genome ID (NCBI database), TaxID (NCBI database), species, genus, phylum, crRNA, tracrRNA, crispr repeat sequence, effector protein length, effector amino acid sequence, etc. See Appendix II for details.

The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

Embodiment 1

According to the microbial genome database, the microorganisms in the human intestinal flora were analyzed, the Cas protein sequence and Crispr sequence were predicted, and all protein sequences 20kb upstream and downstream of Crispr were determined. Then, the protein database in NCBI was compared to obtain homologous proteins with known Type II or Type V proteins. These homologous proteins were analyzed to determine the conserved sites of the key domains of the homologous proteins and the integrity of the proteins, thereby obtaining the Cas protein sequences in Appendix I and Appendix II and the nearby Crispr sequences. The analysis method is shown in Figure 9. These new Crispr-Cas systems belong to the new Type II and Type V Crispr-Cas systems, which have gene editing capabilities different from the existing SpCas9 protein. These new Crispr-Cas systems enrich the existing Crispr-cas systems and can be used in different cells, such as animal cells and plant cells, as needed to play a gene editing function.

Taking BES1 obtained from human intestinal bacteria Veillonella sp AF13-2 (abbreviated as: AF13-2) as an example, it has lower PAM specificity and smaller protein than the existing commercial SpCas9 and LbCpf1. The PAM sequence preference of BES1 is shown in Figure 1. Only when the fourth position is T, the probability of being cut is extremely low (less than 0.05). The PAM sequence of BES1 is NNNV (where V represents bases A, G, C).

Appendix I is a new Cas9 system table, including the strain name, Genome ID (NCBI database), TaxID (NCBI database), species, genus, phylum information, and crRNA, tracrRNA, crispr repeat sequence, effector protein length, effector amino acid sequence. The Cas protein shown in Appendix I has shown the corresponding crRNA, tracrRNA and/or crispr repeat sequence, and those skilled in the art can directly apply it according to the sequence shown.

Appendix II is a new Cpf1 system table, including the strain name, Genome ID (NCBI database), TaxID (NCBI database), species, genus, phylum information, and crRNA, tracrRNA, crispr repeat sequence, effector protein length, effector amino acid sequence of the Crispr-Cas system or Cas protein. The Cas protein shown in Appendix II does not show the corresponding crRNA, tracrRNA and/or crispr repeat sequence. The crRNA, tracrRNA and/or crispr repeat sequence that can help these Cas proteins perform editing functions can be found based on the information of the corresponding Cas protein.

Example 2 Experiment of expressing and purifying BES1 protein

1. Construction of BES1 expression vector

The expression vector was constructed by the In-fusion method. The pET28a vector was digested at two sites, NdeI and EcoR I, and the gene sequence encoded by BES1 was inserted into the cloning region of the vector pET 28a. The six His at the N-terminus of the recombinant BES1 protein amino acid sequence were used as purification tags, and the screening tag was kanamycin. The constructed vector was named pET28a-BES1.

2. Cultivation and induction of BES1 strain

LB liquid medium: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L.

The recombinant expression vector pET 28a-BES1 was transformed into the Escherichia coli expression strain Ecoli.BL21 (DE3), and the bacterial solution was evenly spread on a LB solid medium plate with a kanamycin concentration of 50 μg/mL, and cultured at 37°C overnight. A single colony was picked and cultured in 5 ml LB medium (containing 50 μg/mL kanamycin) at 37°C, 200 rpm, overnight. The bacterial solution obtained above was inoculated into 50 ml LB medium (containing 50 μg/mL kanamycin) at a ratio of 1:100 and cultured at 37°C, 200 rpm, for 4 hours. The expanded culture solution was inoculated into 2L LB liquid medium (containing 50μg/mL kanamycin) at a ratio of 1:100, cultured at 37°C, 200rpm, and when the OD600 value reached about 0.6-0.8, IPTG was added to a final concentration of 0.4mM, and cultured overnight at 16°C, 200rpm for about 16-18h. The induction-completed bacterial solution was centrifuged at 10000g to collect the bacterial cells, and the bacterial cells were frozen at -20°C for use.

3. Extraction and purification of BES1 protein

Purification Buffer Preparation:

(1) Ni column affinity chromatography

Buffer A: 50 mM Tris-HCl + 500 mM NaCl + 20 mM imidazole, pH 7.5.

Buffer B elution buffer: 50 mM Tris-HCl + 500 mM NaCl + 500 mM imidazole, pH 7.5.

(2) Ion exchange chromatography

Buffer C equilibrium buffer: 50mM Tris-HCl + 100mM NaCl, pH 7.0.

Buffer D elution buffer: 50 mM Tris-HCl + 1 M NaCl, pH 7.0.

(3) Protein sample diluent

Buffer E diluent: 50 mM Tris-HCl, pH 7.0.

(4) Protein sample 2× storage solution

Buffer F 2× storage solution: 50 mM Tris-HCl + 300 mM NaCl, pH 7.0.

Resuspend the cells at a ratio of 1g of cells to 15ml of Buffer A, and add PMSF at a final concentration of 1mM. Ultrasonicate the cells until the cell solution becomes clear. Centrifuge the broken cells at 4℃, 12000rpm, for 30min, take the supernatant, filter with a 0.22μm filter membrane, and store at 4℃.

The Ni column affinity chromatography column was washed with water for 5CV, washed with Buffer B for 5CV, and equilibrated with Buffer A for 10CV before loading. After loading, equilibrated for 15CV, 15% Buffer B was used to wash away the impurities, and linear elution (15-100% Buffer B, 10CV) was performed. When the UV value was greater than 100mAU, the protein was collected.

The protein collected by the Ni column was diluted 5 times with Buffer E, the Q anion exchange column was washed with water for 5CV, Buffer C was balanced for 5CV, the protein sample was loaded, and the flow-through liquid was collected when the UV value rose. The SP cation exchange column was balanced with Buffer C for 5CV, and the protein sample obtained in the previous step was loaded. After loading, Buffer C was balanced for 15CV, and then the elution buffer Buffer D was linearly eluted (0-100% Buffer D, 10CV) to collect the protein. The collected protein was dialyzed overnight, and the dialysate was 2× storage Buffer. The final protein concentration was 1mg/mL and the glycerol concentration was 50%. As shown in Figure 2, the SDS-PAGE results showed that the purification effect of the fusion protein was very good and the purity was qualified.

The following Examples 3 and 4 take the Cas9 protein BES1BES1 (SEQ ID NO: 1) found in the human intestinal bacteria Veillonella sp AF13-2 as an example to explore the PAM sequence recognized by the protein and its cleavage function for the target substrate in vitro.

Example 3 Experimental study on obtaining BES1 PAM sequence

1. Preparation of guide RNA

First, we designed a double-stranded DNA transcription template for crRNA and tracrRNA-L based on the predicted crRNA and tracrRNA sequences of BES1 in strain AF13-2 (see Appendix I) (see Table 1 below). At the same time, on this basis, we tried to shorten the sequence of the pairing region of crRNA and tracrRNA-L, and connected them with a GAAA connection sequence to form a single DNA chain, i.e. sgRNA-1, and the transcription template sequence of sgRNA-1 is shown in Table 1 below. At the same time, in order to maximize the activity of the original RNA, sgRNA-3 was designed, and its transcription template sequence is shown in Table 1 below. The deoxynucleotide sequences used in Table 1 are all synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform. The sequences shown in Table 1 are all DNA template sequences for transcription of each RNA. The sequences and secondary structures of crRNA+tracrRNA-L, sgRNA-1, and sgRNA-3 are shown in Figure 3.

Table 1 RNA transcription template sequences used in BES1 chip cleavage experiments

The double-stranded DNA template was prepared by DNA polymerase chain reaction using KAPA HiFiTM heat-activated instant mixed solution (Roche). After the reaction, the double-stranded DNA template was purified using a phenol chloroform isoamyl alcohol mixed solution (Aladdin), and the purity of the purified double-stranded DNA template was measured using a Nanodrop TM 2000 spectrometer (Thermo Fisher Scientific), and the concentration of the purified double-stranded DNA template was measured using a Qubit ^TM double-stranded DNA high-sensitivity quantitative kit (Thermo Fisher Scientific) and a Qubit TM 3.0 fluorescence quantifier.

Then, the double-stranded DNA template was used for transcription. During the transcription, according to the instructions of MEGAscript ^™ T7 Transcription Kit, 2 pmol of double-stranded DNA template was added and incubated at 37°C for 12 hours using Bio-rad S1000™ PCR instrument. RNA was purified using phenol chloroform isoamyl alcohol mixture (Aladdin), and the purity and concentration of the purified RNA were measured using Nanodrop™ 2000 spectrometer (ThermoFisher Scientific).

2. Preparation of single-stranded loop of cleaved substrate

A cleavage substrate for the BES1 protein was prepared, wherein the deoxynucleotide sequences used for the cleavage substrate are shown in the following table (Table 2). The deoxynucleotide sequences used in Table 2 were all synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform.

DNA polymerase chain reaction using Heat activated instant use mixture (Roche) was used to prepare the double-stranded substrate to be cleaved (double-stranded substrate). The two nucleotide sequences PAM_AF13-2_2/1 and PAM_AF13-2_2/2 in Table 2 were denatured at 95 degrees Celsius and then renatured as templates, and the two nucleotide sequences PAM_AF13-2_1 and PAM_AF13-2_3 were used as primers for polymerase chain reaction amplification to obtain double-stranded substrates.

The polymerase chain reaction product was recovered using an E.Z.N.A.TM gel recovery kit, and the purity of the recovered product was measured using a Nanodrop TM 2000 spectrometer (Thermo Fisher Scientific), and the concentration was measured using a Qubit TM double-stranded DNA high-sensitivity quantification kit (Thermo Fisher Scientific) and a Qubit TM 3.0 fluorescence quantification instrument.

Table 2 Deoxynucleotide sequences used in the preparation of cleavage substrates

Then, the double-stranded substrate obtained above is used to perform single-stranded cyclization to obtain a single-stranded circular product. The method is as follows:

Using 1 pmol of the double-stranded DNA substrate prepared above, 1×TA buffer (Epicentre), 120 U of T4 DNA ligase (Epicentre), and 10 mM ATP (NEB) at a final concentration, the reaction product system size was 60 μl and incubated at 37° C. for 1 hour using a Bio-rad S1000 ^™ PCR instrument.

Then, the uncircularized PCR products were digested using EXO III (10 U/μl) (purchased from BGI) and EXO I (3 U/μl) (purchased from BGI) and incubated at 37°C for 30 minutes using a Bio-rad S1000 ^™ PCR instrument. The products were purified using 2.5 volumes of AMPure XP (Beckman ^™ ) and the concentration was measured using the Qubit ^™ single-stranded DNA high-sensitivity quantification kit (ThermoFisher Scientific) and the Qubit ^™ 3.0 (Thermo Fisher Scientific) fluorescence quantification instrument.

3. SE51 sequencing

(1) The single-stranded ring was used to prepare nanospheres for the above-mentioned machine. 6 ng of the single-stranded ring product was taken and diluted to 20 μl with nuclease-free pure water (Ambion ^TM ). 20 μl of Make DnB buffer (BGI) was added, mixed and centrifuged, and incubated at 95°C for 1 min, 65°C for 1 min, 40°C for 1 min, and 4°C for 5 min using a Bio-rad S1000TM PCR instrument.

The reaction product was added with 40 μl of make DnB enzyme mix V2.0 (BGI) and 2 μl of make DnB enzyme mixII V2.0 (BGI), mixed and incubated at 30°C for 20 minutes using a Bio-rad S1000TM PCR instrument. After the reaction, the DnB stop buffer (BGI) was mixed and evenly mixed using a flared pipette tip (Axygen). Then, 30 μl of load DnB buffer (BGI) was added and evenly mixed using a flared pipette tip (Axygen). The library was fixed to a BGITMSEQ500 V3.1 chip (BGI) using a BGITMSEQ500 DnB loader (BGI) to obtain a chip to be sequenced.

(2) Using BGI ^™ SEQ500 SE100 sequencing cartridge (BGI), the above chip was subjected to SE51 sequencing using BGI™ SEQ500 sequencer (BGI) to obtain the sequence information and ID number of each nucleic acid sequence.

4. BES1-PAM native strand sequencing

Since the single-stranded DNA obtained by the above sequencing is used, the single-stranded DNA is used to synthesize the complementary strand (i.e., the native strand), and the obtained double-stranded DNA is used for protein cleavage experiments. Including:

(1) After chip sequencing was completed, the new strand generated by the first sequencing was eluted using 100% formamide (Sigma) on a BGI ^™ SEQ500 DnB loader (BGI).

(2) After the chip was eluted, native strand synthesis was performed on a BGI ^™ SEQ500 sequencer (BGI) using dNTP mix 2 (BGI) to obtain double-stranded DNA with a synthetic length of 50 nucleotides. The 51st base was synthesized using dNTP mix 1 (BGI). This step was to add fluorescent dNTPs to the end of the synthetic strand.

(3) After the above steps are completed, the chip is photographed using a BGI ^™ SEQ500 sequencer (BGI) and saved as original image 1 on the sequencer.

(4) BES1 chip digestion reaction. The double-stranded DNA obtained in step (2) was digested with different RNAs. The buffer used in the reaction was spCas9 1× reaction buffer (NEB), 30 μg of RNA (crRNA+tracrRNA-L, sgRNA-1 or sgRNA-3) prepared in step 1 was added, the final concentration of BES1 protein was 0.1 μM, the final volume of the RNase inhibitor (Epicentre) reaction system was 300 μL, and the mixed solution was pumped into the chip using BGI ^TM SEQ500 DnB loader (BGI) and incubated at 37°C for 5 hours.

(5) The chip was washed three times with 300 μl of washing buffer 2 (BGI).

(6) After the above steps are completed, the chip is photographed using a BGITMSEQ500 sequencer (BGI) and saved as original image 2 on the sequencer.

(7) The BGITMSEQ500 sequencer (BGI) was used to perform manual basecalling on the saved original image 1 and original image 2. The fluorescence signals before and after enzyme digestion were compared using the BGITMSEQ500 sequencer (BGI). The PAM sequence of BES1 was analyzed, and SpCas9 was used as a control. The results are shown in Figure 4.

In the results shown in Figure 4, the horizontal axis shows the 7 sites adjacent to the 3' end of the target sequence, and the vertical axis is the proportion of each base in all positive sequences cut. That is, the vertical axis represents the number of sequences cut as the denominator, determines which base is cut at each position, and calculates the proportion of the four bases at each position. From the results shown in Figure 4, it can be seen that compared with SpCas9, the preference of BES1 under the action of Guide RNA with slightly different structures is not much different.

Example 4 In vitro cleavage experiment of BES1

1. Preparation of guide RNA

According to the method of Example 3, the crRNA transcription template, the double-stranded DNA transcription template of tracrRNA-L, and the double-stranded DNA transcription templates of sgRNA-1 and sgRNA-3 were obtained. At the same time, a shorter tracrRNA-S was designed, and sgRNA-2 was designed using the complete crRNA and tracrRNA-S. The transcription template sequences are shown in Table 3 below. The transcription template DNAs were all synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform.

Table 3 Double-stranded DNA transcription template of sgRNA-2

The above DNA template can be used to transcribe the functional RNA shown in Figure 4, including crRNA+tracrRNA-L, sgRNA-1, sgRNA-2 and sgRNA-3 (wherein the target sequence is replaced by N in Figure 4).

Specifically, the method according to the third embodiment includes:

The double-stranded DNA template was prepared by DNA polymerase chain reaction using KAPA HiFiTM heat-activated instant mixed solution (Roche). After the reaction, the double-stranded DNA template was purified using a phenol-chloroform-isoamyl alcohol mixed solution (Aladdin), and the purity of the purified double-stranded DNA template was measured using a Nanodrop TM 2000 spectrometer (Thermo Fisher Scientific), and the concentration of the purified double-stranded DNA template was measured using a Qubit ^TM double-stranded DNA high-sensitivity quantification kit (Thermo Fisher Scientific) and a Qubit TM 3.0 fluorescence quantification instrument.

Then, the double-stranded DNA template was used for transcription. During the transcription, according to the instructions of MEGAscript ^™ T7 Transcription Kit, 2 pmol of double-stranded DNA template was added and incubated at 37°C for 12 hours using Bio-rad S1000™ PCR instrument. RNA was purified using a phenol chloroform isoamyl alcohol mixture (Aladdin), and the purity and concentration of the purified RNA were measured using a Nanodrop™ 2000 spectrometer (Thermo Fisher Scientific).

2. Preparation of cutting substrate

Target site design: Crispr sequences are usually composed of a leader, multiple repeats, and multiple spacers. The leader can usually serve as the promoter of the Crispr sequence, the repeats can form a hairpin structure, and the spacers are usually composed of captured exogenous DNA. Therefore, the original pro-spacer sequence (such as selected-spacer in Figure 5) on the genome sequence of Veillonella sp.AF13-2 strain (NCBIgenome ID: QTMT00000000) was used as the target site sequence.

PAM sequence design: A 7-N PAM library (spacer and PAM sequences in Figure 6 ) was established to facilitate the cleavage of the BES1 protein.

Cutting substrate design: The synthesized PAM library sequence was cloned into the pMD19 vector to form the pMD19-AF13-2-3'PAM library. We amplified an 842bp cutting substrate sequence in this library (see Figure 7, where the cutting substrate sequence is shown in SEQ ID NO:243), the target site position is 402bp-431bp (see Figure 7), and the PAM position is 432bp-438bp (see Figure 7, i.e., the 7 random bases from the 432nd to the 438th position in SEQ ID NO:24, the underlined part), so the products after cutting are all about 400bp. The reason for such design is that when the resolution of gel electrophoresis is not high, the cutting product will form a wider band, so that we can detect whether it is cut.

The 842 bp cleavage substrate sequence is as follows (N represents any base) (SEQ ID NO: 23):

CTGGCCTTTTGCTCCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGC TCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGTTTGCACGCCTGCCGTTCGACGATTGTAGTAGCTCAAAAGGGAACTGCTACCGAA NNNNNNN AATCTCTGGAAGATCCGCGCGTACCGAGTTCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTC TCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGG (SEQ ID NO: 23).

3. Cutting experiment and results

The cleavage system consisted of functional RNA (the four RNAs shown in FIG4 ), cleavage substrate and BES1 input at a final concentration of 100 nM. The cells were incubated at 20° C., 25° C. and 37° C. for 1 hour, respectively. The cleavage products were identified using 2% agarose gel. The cleavage results are shown in FIG8 .

From the results shown in FIG8 , it can be seen that under incubation at 20° C., 25° C. and 37° C., BES1 plus the four functional RNAs in FIG4 can cleave the target substrate.

Example 5 PAM Preference Identification of BES2, BES4 and BES6 Systems

The PAM identification experimental methods and steps of the three systems BES2, BES4 and BES6 are consistent with the above embodiment, and the main steps are as follows:

(1) Preparation of guide RNA

The tracrRNA and crRNA sequences of the BES2 system in the strain Collinsella sp. Marseille-P2666 were obtained by bioinformatics prediction (see Appendix I), and the double-stranded DNA transcription template of the sgRNA connected and integrated by crRNA and tracrRNA was designed. The specific deoxynucleotide sequences are shown in Table 4 below. BES4 and BES6 belong to the Cpf1 homologous system. This system only needs crRNA to guide the effector protein to achieve genome targeted cleavage without the participation of tracrRNA. The crRNA sequences of the two proteins were predicted by bioinformatics, and their double-stranded DNA transcription templates were designed and synthesized. The specific deoxynucleotide sequences are shown in Table 4 below. The deoxynucleotide sequences used in Table 4 were all synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform.

Table 4:

The preparation steps for the expression of double-stranded DNA transcription template guide RNA for BES2 system gRNA, BES4 and BES6 system crRNA shown in Table 4 are the same as those in Example 3.

(2) PAM identification

The PAM sequences of the BES2, BES4 and BES6 systems detected quickly based on the DNB chip are consistent with those in Example 3. The PAM preferences of the three systems are shown in FIG10 .

Example 6 Identification of in vitro cleavage activity of BES2, BES4 and BES6 systems

First, guide RNA sequences of BES2, BES4 and BES6 systems were expressed by in vitro transcription according to Example 3; secondly, the effector proteins of BES2, BES4 and BES6 systems were expressed and purified in accordance with the experimental method in Example 2; finally, substrate preparation and in vitro cleavage were performed in accordance with the experimental method in Example 4. As shown in Figure 11, all three systems have the activity of cleaving double-stranded DNA in vitro.

Example 7 Identification of editing activity of the BES6 system in human cells

(1) Human cell culture

The inventors selected human HEK293T cells as cells for in vivo editing activity testing. HEK293T cells were cultured in DMEM medium and provided with nutrition by fetal bovine serum (FBS).

(2) RNP preparation

For editing of HEK293T cells, we selected the endogenous gene AAVS1 for targeted cutting verification.

The nucleotide sequence of the targeted region of AAVS1 is as follows:

CCCTTGCTCTCTGCTGTGTTGCTGCCCAAGGATGCTCTTTCCGGAGCACTTCCTTC

TCGGCGCTGCACCACGTGATGTCCTCTGAGCGGATCCTCCCCGTGTCTGGGTCCTCTC

CGGGCATCTCTCCTCCCTCACCCAACCCCATGCCGTcTTCACTCGCTGGGTTCCCTTTT

CCTTCTCCTTCTGGGGCCTGTGCCATCTCTCGTTTCTTAGGATGGCCTTCTCCGACGGA

TGTCTCCCTTGCGTCCCGCCTCCCCTTCTTGTAGGCCTGCATCATCACCGTTTTTCTGG

ACAACCCCAAAGTACCCCGTCTCCCTGGCTTtAGcCACCTCTCCATCCTCTTGCTTTCTT

TGCCTGGACACCCCGTTCTCCTGTGGATTCGGGTCACCTCTCACTCCTTTCATTTGGGC

AGCTCCCCTACCCCCCTTACCTCTCTAGTCTGTGCTAGCTCTCCAGCCCCCTGTCATG

GCATCTTCCAGGGGTCCGAGAGCTCAGCTAGTCTTCTTCCTCCAACCCGGGCCCcTAT

GTCCACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTCCCCGAGCTGGGA

CCACCTTATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCC

CCTCCACCCCACAGTGGGGCCACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCC

TTAGGCCTCCTCCTTCCTAGTCTCCTGATATTGGGTCTAACCCCCACCTCCTGTTAGGC

AGATTCCTTATCTGGTGACACACCCCCATTTCCTGGAGCCATCTCTCTCCTTGCCAGAA

CCTCTAAGGTTTGCTTACGATGGAGCCAGAGAGGATCCTGGGAGGGAGAGCTTGGCA

GGGGGTGGGAGGGAAGGGGGGGATGCGTGACCTGCCCGGTTCTCAGTGGCCACCCT

GCGCTACCCTCTCCCAGAACCTGAGCTGCTCTGACGCGGCTGTCTGGTGCGTTTCACT

GATCCTGGTGCTGCAGCTTCCTTACACTTCCCAAGAGGAGAAGCAGTTTGGAAAAAC

AAAATCAGAATAAGTTGGTCCTGAGTTCTAACTTTGGCTCTTCACCTTTCTAGTCCCCA

ATTTATATTGTTCCTCCGTGCGTCAGTTTTACCTGTGAGATAAGGCCAGTAGCCACCCC

CGTCCTGGCAGGGCTGTGGTGAGGAGGGGGGTGTCCGTGTGGAAAACTCCCTTTGTG

AGAATGGTGCGTCCTAGGTGTTCACCAGGTCGTGGCCGCCTCTACTCCCTTTCTCTTTC

TCCATCCTTCTTTCCTTAAAGAGCCCCCAGTGCTATCTGGACATATTCCTCCGCCCAGA

GCAGGGTCCGCTTCCCTAAGGCCCTGCTCTGGGCTTCTGGGTTTGAGTCCTTGCAAGC

CCAGGAGAGCGCTAGCTTCCCTGTCCCCCTTCCTCGTCCACCATCTCATGCCCTGGCT

CTCCTGCCCCTTCCTACA (SEQ ID NO:27).

For this gene, a targeting site was designed, and its double-stranded DNA transcription template was designed and synthesized. The specific deoxynucleotide sequences are shown in Table 5. The deoxynucleotide sequences used in Table 5 were all synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform.

Table 5:

The BES4 and BES6 targeting AAVS1 site sequences shown in Table 5 were transcribed in vitro using ordered oligonucleotides and MEGAshortscriptTM T7 transcription kit (Invitrogen) according to the manufacturer's recommended method to generate guide RNA. The in vitro expression of BES4 and BES6 effector proteins was consistent with Example 2.

(3) RNP transfer into human cells

In a twelve-well plate, 10 pmol of purified effector protein and 0.5 μl of gRNA were added to each well. RNPs were assembled and transfected into HEK293T cells using the Neon ^™ Transfection System Kit and Nucleofectamine (Invitrogen) according to the manufacturer's protocol.

(4) Identification of editing activity

Harvest cells 2-3 days after RNP transfection and perform T7E1 enzyme digestion assay to detect activity. The steps are as follows:

(a) Collect cells: add 200 μl of 0.5 M EDTA (pH 8.0) to each well of a 12-well plate to resuspend the cells;

(b) Genomic DNA extraction: Genomic DNA was extracted using a genomic DNA extraction kit (Tiangen), and the gDNA concentration was measured using Nanodrop;

(c) Targeted region PCR: GXL Prime was used to amplify the target site region from gDNA. The amplification primers are shown in Table 6 below. The deoxynucleotide sequences used were synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform. The PCR purification and gel extraction kit (MN) was used for purification. The cleanliness of the PCR product was analyzed by agarose gel electrophoresis, and the concentration was measured using Nanodrop.

(d) Denaturation and annealing: Use Bio-rad PCR instrument to denature and anneal the purified product from step (c). An equal amount of substrate DNA (about 200-300 ng/rxn, 10 μl reaction system) is added to the T7E1 digestion reaction.

(e) T7E1 digestion: Add 0.2 μl of T7EI nuclease to 10 μl of the sample in step (d) and perform digestion reaction at 37°C for 20 minutes.

(f) Activity detection: After T7E1 completes the cleavage reaction, loading buffer is added and the bands are detected by agarose gel.

Table 6: List of PCR amplification primers

As shown in FIG. 12 , BES6 has editing activity in human cells.

Example 8 Identification of editing activity of the BES4 system in human cells

(1) Human cell culture

The inventors selected human HEK293T cells as cells for in vivo editing activity testing. HEK293T cells were cultured in DMEM medium and provided with nutrition by fetal bovine serum (FBS).

(2) Plasmid preparation

For the editing of HEK293T cells, we selected the endogenous gene HBG for targeted cutting verification.

The nucleotide sequence of the targeted region of HBG is as follows:

CCCTGCTGTGCTCAGATCAATACTCCGTTGTCTAAGTTGCCTCGAGACTAAAGGC

AACAGGGCTGAAACATCTCCTGGACTCACCTTGAAGTTTCTCAGGATCCACATGCAGCT

TGTCACAGTGCAGTTCACTCAGCTGGGCAAAGGTGCCCTTGAGATCATCCAGGTGCTT

TGTGGCATCTCCCAAGGAAGTCAGCACCTTCTTGCCATGTGCCTTGACTTTGGGGTTG

CCCATGATGGCAGAGGCAGAGGACAGGTTGCCAAAGCTGTCAAAGAACCTCTGGGTC

CATGGGTAGACAACCAGGAGCCTGTGAGATTGACAAGAACAGTTTGACAGTCAGAAG

GTGCCAAAATCCTGAGAAGCGACCTGGACTTTTGCCAGGCACAGGGTCCTTCCTTC

CCTCCCTTGTCCTGGTCACCAGAGCCTACCTTCCCAGGGTTTCTCCTCCAGCATCTTCC

ACATTCACCTTGCCCCACAGGCTTGTGATAGTAGCCTTGTCCTCCTCTGTGAAATGACC

CATGGCGTCTGGACTAGGAGCTTATTGATAACCTCAGACGTTCCAGAAGCGAGTGTGT

GGAACTGCTGAAGGGTGCTTCCTTTTATTCTTCATCCCTAGCCAGCCGCCGGCCCCTG

GCCTCACTGGATACTCTAAGACTATTGGTCAAGTTTGCCTTGTCAAGGCTATTGGTCAA

GGCAAGGCTGGCCAACCCATGGGTGGAGTTTAGCCAGGGACCGTTTCAGACAGATAT

TTGCATTGAGATAGTGTGGGGAAGGGGCCCCCAAGAGGATACTGCTAATTTTTTTTATA

GCCTTTGCCTTGTTCCGATTCAGTCATTCCAGTTTTTCTCTAATTTATTCTTCCCTTTAGC

TAGTTTCCTTCTCCCATCATAGAGGATACCAGGACTTCTTTTGTCAGCCGTTTTTTACCT

TCTTGTCTCTAGCTCCAGTGAGGCCTGTAGTTTAAAGCTAAAGCATGTACCAATTTTTG

AAAAGTTCAGGGATTGTGAAATGTGTTTTAGGCATAGGTCCAGGATTTTTGACGGGAC

AAATCTTAGTCTCTTTCAGTTAGCAGTGGTTTCTAAGGA (SEQ ID NO: 32).

For this region, the inventors designed three targets and synthesized the corresponding plasmid sequences.

BES4-HBG-sg01:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGA

GAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACG

TAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTA

TCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAA

AGGACGAAACACCGAATTTCTACTATTGTAGATGCCAGCCTTGCCTTGACCAATAGTTT

TTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTTTAGCGCGTGC

GCCAATTCTGCAGACAAATGGCTCTAGAGGTACCCGTTACATAACTTACGGTAAATGG

CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATA

GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG

TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGG

CCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACAT

CTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTC

TCCCCATCTCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTG

TGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG

GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCG

GCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAA

AGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCT

CCGCCGCCGCCTCGCGCCGCCCGCCCCGCCTCTGACTGACCGCGTTACTCCCACAGG

TGAGCGGGCGGGACGGCCCTTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAG

GGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCT

GAAATCACTTTTTTTCAGGTTGGACCGGTGCCACCATGGACTATAAGGACCACGACGG

AGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAG

AAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCATGCAGGAGAGAAAGAA

GATCAGCCACCTGACCCACAGAAACAGCGTGAAGAAAACCATCAGAATGCAGCTGA

ACCCCTGTGGGAAAGACCATGGACTACTTCCAGGCCAAGCAGATCCTGGAGAACGACG

AGAAGCTGAAGGAGGACTACCAGAAGATCAAGGAGATCGCCGACAGATTCTACAGA

AACCTGAACGAGGACGTGCTGAGCAAAACCGGACTGGACAAGCTGAAGGACTACGC

CGAGATCTACTACCATTGCAACACCGACGCCGACAGAAAGAGACTGAACGAGTGCGC

CAGCGAGCTGAGAAAGGAGATCGTGAAGAACTTCAAGAACAGAGATGAGTACAACA

AGCTGTTCAACAAGAAGATGATCGAGATCGTGCTGCCCAAGCACCTGAAGAACGAGG

ACGAGAAGGAAGGTGGTGGCCAGCTTCAAGAACTTCACCACCTACTTCACCGGCTTCT

TCACCAACAGAAAGAACATGTACAGCGACGGCGAAGAGTCTACCGCTATTGCCTACA

GATGCATCAACGAGAACCTGCCCAAGCACCTGGACAACGTGAAGGTGTTCGAGAAG

GCCATCAGCAAGCTGAGCAAGAACGCCATCGACGACCTGGATGCCACATATTCTGGCC

TGTGCGGCACAAATCTGTACGACGTGTTCACCGTGGACTACTTCAACTTCCTGCTGCC

CCAAAGCGGAATCACCGAGTACAACAAGATCATCGGCGGCTACACAACAAGCGACGG

CACCAAAGTGAAGGGCATCAACGAGTACATCAACCTGTACAACCAGCAGGTGAGCAA

GAGAGACAAGATCCCCAACCTGAAGATCCTGTACAAGCAGATCCTGAGCGAGAGCGA

GAAGGTGTCTTTCATCCCCCCCAAGTTCGAGGACGACAACGAACTGCTGTCTGCCGT

GAGCGAGTTCTATGCCAACGACGAGACATTTGATGGCATGCCCTGAAGAAAGCCATC

GACGAAACCAAACTGCTGTTCGGCAACCTGGACAACAGCAGCCTGAACGGCATCTAC

ATCCAGAACGACAGAAGCGTGACCAACCTGAGCAACAGCATGTTCGGCAGCTGGAG

CGTGATTGAGGACCTGTGGAACAAGAACTACGACAGCGTGAACAGCAACAGCAGAA

TCAAGGACATCCAGAAGAGAGAGGACAAGAGAAAGAAGGCCTACAAGGCCGAGAA

GAAGCTGAGCCTGAGCTTCCTGCAGGTGCTGATCAGCAACAGCGAGAACGACGAGAT

CAGAAAGAAGAGCATCGTGGACTACTACAAGACCAGCCTGATGCAGCTGACCGACAA

CCTGAGCGACAAGTACAAAGAAGCCGCCCCCCTGTTTTCTGAGAACTACGACAACGA

GAAGGGCCTGAAGAACGACGACAAGAGCATCAGCCTGATCAAGAACTTCCTGGACG

CCATCAAGGAGATCGAGAAGTTCATCAAGCCCCTGAGCGAGACAAATATCACCGGCG

AGAAGAACGACCTGTTCTACAGCCAGTTCACCCCCCTGCTGGACAACATCAGCAGAA

TCGACAGACTGTACGACAAGGTGAGAAAACTACGTGACCCAGAAGCCCTTCAGCACCG

ACAAGATCAAGCTGAACTTCGGCAACAGCCAGCTTCTGAACGGCTGGGACAGAAAC

AAGGAGAAGGACTGTGGCGCTGTGCTGCTGTAAGGACGAGAAGTACTACCTGGCC

ATCATCGACAAGAGCAACAACAGCATCCTGGAGAACATCGACTTCCAGGACTGCAAC

GAGAGCGACTACTACGAGAAGATCGTGTACAAGCTGCTGACCAAGATCTCTGGCAAC

CTGCCCAGAGGTGTTCTTCAGCGAGAAGCACAAGAAGCTGCTGAGCCCCAGCGATGAG

ATCCTGAAGATCTACAAGAGCGGCACCTTCAAGAAGGGCGACAAGTTCAGCCTTGAC

GACTGCCACAAGCTGATCGACTTCTACAAGGAGAGCTTCAAGAAGTACCCCAAGTGG

CTGATCTACAACTTCAAGTTCAAGAACACCAACGAGTACAACGACATCAGCGAGTTCT

ACAACGACGTGGCCAGCCAGGGATACAACATCAGCAAGATGAAGATCCCCACCAGCT

TCATCGACAAGCTGGTGGACGAGGGCAAGATCTACCTGTTCCAGCTGTACAACAAGG

ACTTCAGCCCCCACAGCAAGGGAACACCTAACCTGCACAACCCTGTACTTCAAGATGC

TGTTCGACGAGAGAAACCTGGAGGACGTGGTGTACAAGCTGAATGGCGAGGCCGAG

ATGTTTTACAGACCCGCCAGCATCAAGTATGACAAGCCCACCCACCCTAAGAACACCC

CCATCAAGAACAAGAACACCCTGAACGACAAGAAGGCCAGCACCTTCCCTTACGACC

TGATCAAGGACAAGAGATACACCAAGTGGCAGTCAGCCTGCACTTCCCCATCACCAT

GAACTTCAAGGCCCCCGACAGAGCCATGATCAACGACGACGTGAGAAACCTGCTGA

AGAGCTGCAACAACAACTTCATCATCGGCATCGACAGAGGCGAGAGAAACCTGCTGT

ACGTGAGCGTGATCGATAGCAACGGCGCCATCATCTACCAGCACAGCCTGAACATCAT

CGGCAACAAGTTCAAGGGCAAGACCTACGAAAACCAACTACAGAGAGAAGCTGGCCA

CCAGAGAGAAGGAGAGAACCGAGCAGAGAAGAAACTGGAAGGCCATCGAGAGCAT

CAAGGAGCTGAAGGAGGGCTACATCAGCCAAACCGTGCACGTGATTTGCCAGCTGGT

GGTGAAGTACGACGCCATCATCGTGATGGAGAAGCTGACCGACGGCTTCAAGAGAGG

CAGAACCAAGTTCGAGAAGCAGGTGTACCAGAAGTTCGAGAAGATGCTGATCGACA

AGCTGAACTACTACGTGGACAAGAAGCTGGACCCCAATGAGGAAGGCGGACTGCTG

CATGCTTATCAGCTGACCAACAAGCTGGACAGCTTCGACAAGCTGGGAATGCAGAGC

GGCTTCATCTTCTACGTCAGACCCGACTTCACCAGCAAAATCGACCCCGTGACCGGAT

TTGTGAACCTGCTGTACCCCAGATACGAGAACATCGACAAGGCCAAGGACATGATCA

GCAGATTCGACGACATCAGATACAACGCCGGCGAGGACTTCTTCGAGTTCGACATCG

ACTACGACAAGTTCCCCAAGACCGCCAGCGACTACAGAAAGAAGTGGACCATCTGCA

CCAACGGCGAGAGAATCGAGGCCTTCAGAAACCCCGCCAACAACAACGAGTGGAGC

TACAGAACCATCATCCTGGCCGAGAAGTTCAAGGAGCTGTTCGACAACAACAGCATC

AACTACAGAGACAGCGACGACCTGAAAGCCGAGATCCTGAGCCAAACCAAGGGCAA

GTTCTTCGAGGACTTCTTCAAGCTGCTGAGACTGACCCTGCAGATGAGAAACAGCAA

CCCCGAAACCGGAGAGGACAGGATTCTGAGCCCCGTGAAGGACAAGAACGGCAACT

TCTACGACAGCAGCAAGTACGACGAGAAGAGCAAGCTGCCCTGTGACGCTGATGCTA

ACGGCGCTTACAACATCGCCAGAAAGGGCCTGTGGATCGTGGAGCAGTTCAAGAAGG

CCGACAACGTGTCTGCTGTGGAACCCGTGATCCACAACGACAAGTGGCTGAAGTTCG

TGCAGGAGAACGACATGGCCAACAACAAAAAGGCCGGCGGCCACGAAAAAGGCCGG

CCAGGCAAAAAAGAAAAAGGAATTCGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAA

CATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTC

ACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTC

AGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTC

ATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCT

ACGGCGTGCAGTGCTTCAGCCGCTACCCGACCACATGAAGCAGCACGACTTCTTCA

AGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACG

GCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGC

ATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTG

GAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGC

ATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC

GACCACTACCAGCAGAACACCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC

CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCAC

ATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGT

ACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAG

CCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCA

CTGTCCTTTCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCT

ATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAG

CAGGCATGCTGGGGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC

TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG

CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCT

GATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCA

ACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC

AGCGTGACCGCTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTTCGCTTTCTTCCCTTC

CTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGGCTCCCTTTA

GGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATG

GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTC

CACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCTCG

GGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAG

CTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGG

TGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGC

CAACACCCGCTGACGCGCCCTGACGGCTTGTCTGCTCCCGGCATCCGCTTACAGAC

AAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGA

AACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATA

ATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTA

TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGAT

AAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC

CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGT

GAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGA

TCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATG

AGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGA

GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTC

ACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA

CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGG

AGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGA

ACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGC

AATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGG

CAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGG

CCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGAAGCCG

CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA

CGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG

CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG

ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCA

TGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCGTAGAAAA

GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA

AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT

TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG

CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC

TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGA

CTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG

CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA

GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGCAGGTATCCGGTAAG

CGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC

TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT (SEQ ID NO: 33).

BES4-HBG-sg02:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAATTTCTACTATTGTAGATA CCAATAGCCTTGACAAGGCAAATT

TTTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTTTAGCGCGTG

CGCCAATTCTGCAGACAAATGGCTCTAGAGGTACCCGTTACATAACTTACGGTAAATG

GCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAAT

AGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA

GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATG

GCCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGGTACA

TCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACT

CTCCCCATCTCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTT

GTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCCAGGCGGGGCGGGGCG

GGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGC

GGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAA

AAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCG

CTCCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCCTTGACTGACCGCGTTACTCCCACAG

GTGAGCGGGCGGGACGGCCCTTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAA

GGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCC

TGAAATCACTTTTTTTCAGGTTGGACCGGTGCCACCATGGACTATAAGGACCACGACG

GAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAA

GAAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCATGCAGGAGAGAAAGA

AGATCAGCCACCTGACCCACAGAAACAGCGTGAAGAAAACCATCAGAATGCAGCTG

AACCCCGTGGGAAAGACCATGGACTACTTCCAGGCCAAGCAGATCCTGGAGAACGAC

GAGAAGCTGAAGGAGGACTACCAGAAGATCAAGGAGATCGCCGACAGATTCTACAG

AAACCTGAACGAGGACGTGCTGAGCAAAACCGGACTGGACAAGCTGAAGGACTACG

CCGAGATCTACTACCATTGCAACACCGACGCCGACAGAAAGAGACTGAACGAGTGCG

CCAGCGAGCTGAGAAAGGAGATCGTGAAGAACTTCAAGAACAGAGATGAGTACAAC

AAGCTGTTCAACAAGAAGATGATCGAGATCGTGCTGCCCAAGCACCTGAAGAACGAG

GACGAGAAGGAAGTGGTGGCCAGCTTCAAGAACTTCACCACCTACTTCACCGGCTTC

TTCACCAACAGAAAGAACATGTACAGCGACGGCGAAGAGTCTACCGCTATTGCCTAC

AGATGCATCAACGAGAACCTGCCCAAGCACCTGGACAACGTGAAGGTGTTCGAGAA

GGCCATCAGCAAGCTGAGCAAGAACGCCATCGACGACCTGGATGCCACATATTCTGG

CCTGTGCGGCACAAATCTGTACGACGTGTTCACCGTGGACTACTTCAACTTCCTGCTG

CCCCAAAGCGGAATCACCGAGTACAACAAGATCATCGGCGGCTACACAACAAGCGAC

GGCACCAAAGTGAAGGGCATCAACGAGTACATCAACCTGTACAACCAGCAGGTGAGC

AAGAGAGACAAGATCCCCAACCTGAAGATCCTGTACAAGCAGATCCTGAGCGAGAGC

GAGAAGGTGTCTTTCATCCCCCCCAAGTTCGAGGACGACAACGAACTGCTGTCTGCC

GTGAGCGAGTTCTATGCCAACGACGAGACATTTGATGGCATGCCCTGAAGAAAGCC

ATCGACGAAACCAAACTGCTGTTCGGCAACCTGGACAACAGCAGCCTGAACGGCATC

TACATCCAGAACGACAGAAGCGTGACCAACCTGAGCAACAGCATGTTCGGCAGCTGG

AGCGTGATTGAGGACCTGTGGAACAAGAACTACGACAGCGTGAACAGCAACAGCAG

AATCAAGGACATCCAGAAGAGAGAGGACAAGAGAAAGAAGGCCTACAAGGCCGAG

AAGAAGCTGAGCCTGAGCTTCCTGCAGGTGCTGATCAGCAACAGCGAGAACGACGA

GATCAGAAAGAAGAGCATCGTGGACTACTACAAGACCAGCCTGATGCAGCTGACCGA

CAACCTGAGCGACAAGTACAAAGAAGCCGCCCCCCTGTTTTCTGAGAACTACGACAA

CGAGAAGGGCCTGAAGAACGACGACAAGAGCATCAGCCTGATCAAGAACTTCCTGG

ACGCCATCAAGGAGATCGAGAAGTTCATCAAGCCCCTGAGCGAGACAAATATCACCG

GCGAGAAGAACGACCTGTTCTACAGCCAGTTCACCCCCCTGCTGGACAACATCAGCA

GAATCGACAGACTGTACGACAAGGTGAGAAAACTACGTGACCCAGAAGCCCTTCAGCA

CCGACAAGATCAAGCTGAACTTCGGCAACAGCCAGCTTCTGAACGGCTGGGACAGA

AACAAGGAGAAGGACTGTGGCGCTGTGCTGCTGTAAGGACGAGAAGTACTACCTG

GCCATCATCGACAAGAGCAACAACAGCATCCTGGAGAACATCGACTTCCAGGACTGC

AACGAGAGCGACTACTACGAGAAGATCGTGTACAAGCTGCTGACCAAGATCTCTGGC

AACCTGCCCAGAGTGTTCTTCAGCGAGAAGCACAAGAAGCTGCTGAGCCCCAGCGAT

GAGATCCTGAAGATCTACAAGAGCGGCACCTTCAAGAAGGGCGACAAGTTCAGCCTT

GACGACTGCCACAAGCTGATCGACTTCTACAAGGAGAGCTTCAAGAAGTACCCCAAG

TGGCTGATCTACAACTTCAAGTTCAAGAACACCAACGAGTACAACGACATCAGCGAG

TTCTACAACGACGTGGCCAGCCAGGGATACAACATCAGCAAGATGAAGATCCCCACC

AGCTTCATCGACAAGCTGGTGGACGAGGGCAAGATCTACCTGTTCCAGCTGTACAAC

AAGGACTTCAGCCCCCACAGCAAGGGAACACCTAACCTGCACACCCTGTACTTCAAG

ATGCTGTTCGACGAGAGAAACCTGGAGGACGTGGTGTACAAGCTGAATGGCGAGGCC

GAGATGTTTTACAGACCCGCCAGCATCAAGTATGACAAGCCCACCCACCCTAAGAAC

ACCCCCATCAAGAACAAGAACACCCTGAACGACAAGAAGGCCAGCACCTTCCCCTAC

GACCTGATCAAGGACAAGAGATACACCAAGTGGCAGTCAGCCTGCACTTCCCCATC

ACCATGAACTTCAAGGCCCCCGACAGAGCCATGATCAACGACGACGTGAGAAACCTG

CTGAAGAGCTGCAACAACAACTTCATCATCGGCATCGACAGAGGCGAGAGAAACCTG

CTGTACGTGAGCGTGATCGATAGCAACGGCGCCATCATCTACCAGCACAGCCTGAACA

TCATCGGCAACAAGTTCAAGGGCAAGACCTACGAAAACCAACTACAGAGAGAAGCTG

GCCACCAGAGAGAAGGAGAGAACCGAGCAGAGAAGAAACTGGAAGGCCATCGAGA

GCATCAAGGAGCTGAAGGAGGGCTACATCAGCCAAACCGTGCACGTGATTTGCCAGC

TGGTGGTGAAGTACGACGCCATCATCGTGATGGAGAAGCTGACCGACGGCTTCAAGA

GAGGCAGAACCAAGTTCGAGAAGCAGGTGTACCAGAAGTTCGAGAAGATGCTGATC

GACAAGCTGAACTACTACGTGGACAAGAAGCTGGACCCCAATGAGGAAGGCGGACT

GCTGCATGCTTATCAGCTGACCAACAAGCTGGACAGCTTCGACAAGCTGGGAATGCA

GAGCGGCTTCATCTTCTACGTCAGACCCGACTTCACCAGCAAAATCGACCCGTGACC

GGATTTGTGAACCTGCTGTACCCCAGATACGAGAACATCGACAAGGCCAAGGACATG

ATCAGCAGATTCGACGACATCAGATACAACGCCGGCGAGGACTTCTTCGAGTTCGACA

TCGACTACGACAAGTTCCCCAAGACCGCCAGCGACTACAGAAAGAAGTGGACCATCT

GCACCAACGGCGAGAGAATCGAGGCCTTCAGAAAACCCCGCCAACAACAACGAGTGG

AGCTACAGAACCATCATCCTGGCCGAGAAGTTCAAGGAGCTGTTCGACAACAACAGC

ATCAACTACAGAGACAGCGACGACCTGAAAGCCGAGATCCTGAGCCAAACCAAGGG

CAAGTTCTTCGAGGACTTCTTCAAGCTGCTGAGACTGACCCTGCAGATGAGAAACAG

CAACCCCGAAACCGGAGAGGACAGGATTCTGAGCCCCGTGAAGGACAAGAACGGCA

ACTTCTACGACAGCAGCAAGTACGACGAGAAGAGCAAGCTGCCCTGTGACGCTGATG

CTAACGGCGCTTACAACATCGCCAGAAAGGGCCTGTGGATCGTGGAGCAGTTCAAGA

AGGCCGACAACGTGTCTGCTGTGGAACCCGTGATCCACAACGACAAGTGGCTGAAGT

TCGTGCAGGAGAACGACATGGCCAACAACAAAAGGCCGGCGGCCACGAAAAAGGCC

GGCCAGGCAAAAAAGAAAAAGGAATTCGGCAGTGGAGAGGGCAGAGGAAGTCTGCT

AACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGT

TCACCGGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT

TCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGT

TCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA

CCTACGGCGTGCAGTGCTTCAGCCGCTACCCGACCACATGAAGCAGCACGACTTCTT

CAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGA

CGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACC

GCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAG

CTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC

GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACCGGCAGCGTGCAGCTC

GCCGACCACTACCAGCAGAACACCCCATCGGCGACGGCCCCGTGCTGCTGCCCGAC

AACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGAT

CACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAG

CTGTACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTG

CCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACT

CCCACTGTCCTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCA

TTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGA

ATAGCAGGCATGCTGGGGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTC

CCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCC

CGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGG

CGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCA

AAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT

ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCT

TCCCTTCCTTTCTCGCCACGTTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTC

CCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGG

GTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTT

GGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCT

ATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTTAAAA

AATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAAT

TTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGAC

ACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTA

CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATC

ACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTC

ATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAA

CCCCTATTTGTTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACC

CTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG

TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACG

CTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA

CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAA

TGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCAC

CAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC

CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCG

AAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT

GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTG

TAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTC

CCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCG

CTCGGCCCTTCCGGCTGGCTGGTTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGA

AGCCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTAT

CTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGAT

AGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTT

AGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATA

ATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT

AGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC

AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAA

CTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCT

AGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTC

GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG

GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGG

GTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTAC

AGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGCGGGACAGGTAT

CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAA

ACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT

TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT(SEQ ID NO:34)

BES4-HBG-SG03:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAATTTCTACTATTG TAGATCCTTGTCAAGGCTATTGGTCAAGTTTTTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTTTAGCGCGTGCGCCAATTCTGCAGACAAATGGCTCTAGAGGTACCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTG GCAG

TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGG

CCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACAT

CTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTC

TCCCCATCTCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTG

TGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG

GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCG

GCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAA

AGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCT

CCGCCGCCGCCTCGCGCCGCCCGCCCCGCCTCTGACTGACCGCGTTACTCCCACAGG

TGAGCGGGCGGGACGGCCCTTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAG

GGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCT

GAAATCACTTTTTTTCAGGTTGGACCGGTGCCACCATGGACTATAAGGACCACGACGG

AGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAG

AAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCATGCAGGAGAGAAAGAA

GATCAGCCACCTGACCCACAGAAACAGCGTGAAGAAAACCATCAGAATGCAGCTGA

ACCCCTGTGGGAAAGACCATGGACTACTTCCAGGCCAAGCAGATCCTGGAGAACGACG

AGAAGCTGAAGGAGGACTACCAGAAGATCAAGGAGATCGCCGACAGATTCTACAGA

AACCTGAACGAGGACGTGCTGAGCAAAACCGGACTGGACAAGCTGAAGGACTACGC

CGAGATCTACTACCATTGCAACACCGACGCCGACAGAAAGAGACTGAACGAGTGCGC

CAGCGAGCTGAGAAAGGAGATCGTGAAGAACTTCAAGAACAGAGATGAGTACAACA

AGCTGTTCAACAAGAAGATGATCGAGATCGTGCTGCCCAAGCACCTGAAGAACGAGG

ACGAGAAGGAAGGTGGTGGCCAGCTTCAAGAACTTCACCACCTACTTCACCGGCTTCT

TCACCAACAGAAAGAACATGTACAGCGACGGCGAAGAGTCTACCGCTATTGCCTACA

GATGCATCAACGAGAACCTGCCCAAGCACCTGGACAACGTGAAGGTGTTCGAGAAG

GCCATCAGCAAGCTGAGCAAGAACGCCATCGACGACCTGGATGCCACATATTCTGGCC

TGTGCGGCACAAATCTGTACGACGTGTTCACCGTGGACTACTTCAACTTCCTGCTGCC

CCAAAGCGGAATCACCGAGTACAACAAGATCATCGGCGGCTACACAACAAGCGACGG

CACCAAAGTGAAGGGCATCAACGAGTACATCAACCTGTACAACCAGCAGGTGAGCAA

GAGAGACAAGATCCCCAACCTGAAGATCCTGTACAAGCAGATCCTGAGCGAGAGCGA

GAAGGTGTCTTTCATCCCCCCCAAGTTCGAGGACGACAACGAACTGCTGTCTGCCGT

GAGCGAGTTCTATGCCAACGACGAGACATTTGATGGCATGCCCTGAAGAAAGCCATC

GACGAAACCAAACTGCTGTTCGGCAACCTGGACAACAGCAGCCTGAACGGCATCTAC

ATCCAGAACGACAGAAGCGTGACCAACCTGAGCAACAGCATGTTCGGCAGCTGGAG

CGTGATTGAGGACCTGTGGAACAAGAACTACGACAGCGTGAACAGCAACAGCAGAA

TCAAGGACATCCAGAAGAGAGAGGACAAGAGAAAGAAGGCCTACAAGGCCGAGAA

GAAGCTGAGCCTGAGCTTCCTGCAGGTGCTGATCAGCAACAGCGAGAACGACGAGAT

CAGAAAGAAGAGCATCGTGGACTACTACAAGACCAGCCTGATGCAGCTGACCGACAA

CCTGAGCGACAAGTACAAAGAAGCCGCCCCCCTGTTTTCTGAGAACTACGACAACGA

GAAGGGCCTGAAGAACGACGACAAGAGCATCAGCCTGATCAAGAACTTCCTGGACG

CCATCAAGGAGATCGAGAAGTTCATCAAGCCCCTGAGCGAGACAAATATCACCGGCG

AGAAGAACGACCTGTTCTACAGCCAGTTCACCCCCCTGCTGGACAACATCAGCAGAA

TCGACAGACTGTACGACAAGGTGAGAAAACTACGTGACCCAGAAGCCCTTCAGCACCG

ACAAGATCAAGCTGAACTTCGGCAACAGCCAGCTTCTGAACGGCTGGGACAGAAAC

AAGGAGAAGGACTGTGGCGCTGTGCTGCTGTAAGGACGAGAAGTACTACCTGGCC

ATCATCGACAAGAGCAACAACAGCATCCTGGAGAACATCGACTTCCAGGACTGCAAC

GAGAGCGACTACTACGAGAAGATCGTGTACAAGCTGCTGACCAAGATCTCTGGCAAC

CTGCCCAGAGGTGTTCTTCAGCGAGAAGCACAAGAAGCTGCTGAGCCCCAGCGATGAG

ATCCTGAAGATCTACAAGAGCGGCACCTTCAAGAAGGGCGACAAGTTCAGCCTTGAC

GACTGCCACAAGCTGATCGACTTCTACAAGGAGAGCTTCAAGAAGTACCCCAAGTGG

CTGATCTACAACTTCAAGTTCAAGAACACCAACGAGTACAACGACATCAGCGAGTTCT

ACAACGACGTGGCCAGCCAGGGATACAACATCAGCAAGATGAAGATCCCCACCAGCT

TCATCGACAAGCTGGTGGACGAGGGCAAGATCTACCTGTTCCAGCTGTACAACAAGG

ACTTCAGCCCCCACAGCAAGGGAACACCTAACCTGCACAACCCTGTACTTCAAGATGC

TGTTCGACGAGAGAAACCTGGAGGACGTGGTGTACAAGCTGAATGGCGAGGCCGAG

ATGTTTTACAGACCCGCCAGCATCAAGTATGACAAGCCCACCCACCCTAAGAACACCC

CCATCAAGAACAAGAACACCCTGAACGACAAGAAGGCCAGCACCTTCCCTTACGACC

TGATCAAGGACAAGAGATACACCAAGTGGCAGTCAGCCTGCACTTCCCCATCACCAT

GAACTTCAAGGCCCCCGACAGAGCCATGATCAACGACGACGTGAGAAACCTGCTGA

AGAGCTGCAACAACAACTTCATCATCGGCATCGACAGAGGCGAGAGAAACCTGCTGT

ACGTGAGCGTGATCGATAGCAACGGCGCCATCATCTACCAGCACAGCCTGAACATCAT

CGGCAACAAGTTCAAGGGCAAGACCTACGAAAACCAACTACAGAGAGAAGCTGGCCA

CCAGAGAGAAGGAGAGAACCGAGCAGAGAAGAAACTGGAAGGCCATCGAGAGCAT

CAAGGAGCTGAAGGAGGGCTACATCAGCCAAACCGTGCACGTGATTTGCCAGCTGGT

GGTGAAGTACGACGCCATCATCGTGATGGAGAAGCTGACCGACGGCTTCAAGAGAGG

CAGAACCAAGTTCGAGAAGCAGGTGTACCAGAAGTTCGAGAAGATGCTGATCGACA

AGCTGAACTACTACGTGGACAAGAAGCTGGACCCCAATGAGGAAGGCGGACTGCTG

CATGCTTATCAGCTGACCAACAAGCTGGACAGCTTCGACAAGCTGGGAATGCAGAGC

GGCTTCATCTTCTACGTCAGACCCGACTTCACCAGCAAAATCGACCCCGTGACCGGAT

TTGTGAACCTGCTGTACCCCAGATACGAGAACATCGACAAGGCCAAGGACATGATCA

GCAGATTCGACGACATCAGATACAACGCCGGCGAGGACTTCTTCGAGTTCGACATCG

ACTACGACAAGTTCCCCAAGACCGCCAGCGACTACAGAAAGAAGTGGACCATCTGCA

CCAACGGCGAGAGAATCGAGGCCTTCAGAAACCCCGCCAACAACAACGAGTGGAGC

TACAGAACCATCATCCTGGCCGAGAAGTTCAAGGAGCTGTTCGACAACAACAGCATC

AACTACAGAGACAGCGACGACCTGAAAGCCGAGATCCTGAGCCAAACCAAGGGCAA

GTTCTTCGAGGACTTCTTCAAGCTGCTGAGACTGACCCTGCAGATGAGAAACAGCAA

CCCCGAAACCGGAGAGGACAGGATTCTGAGCCCCGTGAAGGACAAGAACGGCAACT

TCTACGACAGCAGCAAGTACGACGAGAAGAGCAAGCTGCCCTGTGACGCTGATGCTA

ACGGCGCTTACAACATCGCCAGAAAGGGCCTGTGGATCGTGGAGCAGTTCAAGAAGG

CCGACAACGTGTCTGCTGTGGAACCCGTGATCCACAACGACAAGTGGCTGAAGTTCG

TGCAGGAGAACGACATGGCCAACAACAAAAAGGCCGGCGGCCACGAAAAAGGCCGG

CCAGGCAAAAAAGAAAAAGGAATTCGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAA

CATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAGGGCGAGGAGCTGTTC

ACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTC

AGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTC

ATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCT

ACGGCGTGCAGTGCTTCAGCCGCTACCCGACCACATGAAGCAGCACGACTTCTTCA

AGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACG

GCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGC

ATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTG

GAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGC

ATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC

GACCACTACCAGCAGAACACCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC

CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCAC

ATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGT

ACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAG

CCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCA

CTGTCCTTTCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCT

ATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAG

CAGGCATGCTGGGGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC

TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG

CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCT

GATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCA

ACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC

AGCGTGACCGCTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTTCGCTTTCTTCCCTTC

CTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGGCTCCCTTTA

GGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATG

GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTC

CACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCTCG

GGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAG

CTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGG

TGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGC

CAACACCCGCTGACGCGCCCTGACGGCTTGTCTGCTCCCGGCATCCGCTTACAGAC

AAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGA

AACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATA

ATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTA

TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGAT

AAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC

CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGT

GAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGA

TCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATG

AGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGA

GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTC

ACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA

CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGG

AGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGA

ACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGC

AATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGG

CAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGG

CCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGAAGCCG

CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA

CGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG

CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG

ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCA

TGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCGTAGAAAA

GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA

AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT

TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG

CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC

TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGA

CTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG

CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA

GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGCAGGTATCCGGTAAG

CGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC

TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT (SEQ ID NO: 35).

PX458-HBG-SG01:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCCTTGTCAAGGCTATTGGTC AGTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGTTTTAGAGCTAGAAATAGCAAGTTAAAA TAAGGCTAGTCCGTTTTTAGCGCGTGCGCCAATTCTGCAGACAAATGGCTCTAGAGGTACCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCC GCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCC

CCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGC

AGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCCAGGCGGGGCGGGGCGGGGC

GAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCG

CGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGC

GAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCG

CCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGA

GCGGGCGGGACGGCCCTTTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGT

TTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAA

ATCACTTTTTTTCAGGTTGGACCGGTGCCACCATGGACTATAAGGACCACGACGGAGA

CTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAG

AAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGG

CCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGT

GCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGA

ACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGA

AGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAA

GAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTTCTTCCACAGACTGGAA

GAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAAC

ATCGTGGACGAGGTGGCCTACCACGAGAAGTAACCCACCATCTACCACCTGAGAAAG

AAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCC

CACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC

AGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAG

GAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTG

AGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAA

TGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAG

CAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGA

CGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGC

CGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGA

GATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCA

GGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGA

GATTTTCTTCGACCAGAGCAAGAACGGGCTACGCCGGCTACATTGACGGCGGAGCCAG

CCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGA

GGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCG

ACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGC

GGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCC

TGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGC

CTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGG

TGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGA

ACCTGCCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCG

TGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCT

TCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGG

AAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGA

CTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCAC

GATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGAC

ATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGG

AACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGC

GGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGG

GACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAAC

AGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAG

AAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCC

GGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTC

GTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGA

GAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATC

GAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAA

CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATAT

GTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATC

GTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGC

GACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGAT

GAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGA

CAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCAT

CAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGG

ACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAG

TGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAA

AGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGT

GGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGA

CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCA

AGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGAT

TACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAAC

CGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAG

CATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAA

AGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTG

GGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTG

GTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCT

GGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGA

AGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTC

CCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCA

GAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAG

CCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGT

GGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAA

GAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCA

CCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACC

AATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGG

TACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGC

CTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGC

CACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGAATTCGGCAGTGGAGAGGGC

AGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGTGAGCAAG

GGCGAGGAGCTGTTCACCGGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTA

AACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAA

GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT

CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAA

GCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCAT

CTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG

ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACA

TCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGA

CAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACG

GCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCG

TGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCA

ACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTC

TCGGCATGGACGAGCTGTACAAGGAATTCTAACTAGAGCTCGCTGATCAGCCTCGACT

GTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCT

GGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT

CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAG

GATTGGGAAGAGAATAGCAGGCATGCTGGGGAGCGGCCGCAGGAACCCCTAGTGATG

GAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAG

GTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

CTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCAC

ACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGC

GGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAGCGCCCGC

TCCTTTCGCTTTCTTCCCTTCCTTTCTCCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTC

TAAATCGGGGCTCCCTTTAGGGTCCGATTTAGTGCTTTACGGCACCTCGACCCCAA

AAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTT

CGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAA

CAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGG

TCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATA

TTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTA

AGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTC

CCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGG

TTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTT

TATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGA

AATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTC

ATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTAT

TCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGGCATTTTGCCTTCCTGTTTTTGC

TCACCCAAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGT

GGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAA

GAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCG

TATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG

GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAA

TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAA

CGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA

CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTG

ACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACT

ACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCA

GGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTTATTGCTGATAAATCTGGAG

CCGGTGAGCGTGGAAGCCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGGAGTCAGGCAACTATGGATGAACGAAATAG

ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTT

TACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTG

AAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTG

AGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGC

GTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG

GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC

CAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGC

ACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA

AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC

GGGCTGAACGGGGGGTTTCGTGCACAGCCCAGCTTGGAGCGAACGACCTACACCG

AACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA

AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAG

CTTCCAGGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT

TGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT (SEQ ID NO: 36).

After obtaining the commercially synthesized plasmid and strain required for the BES4 activity detection experiment, amplify them by direct inoculation:

(a) Take 15 mL of antibiotic-free LB liquid medium, add 15 μL of 1000X Amp antibiotic, then use a white pipette tip to pick up the strain containing the target plasmid, put it into the medium, and culture it at 37°C, 200 rpm overnight;

(b) Centrifuge the overnight cultured strain at 8000 rpm for 3 min, centrifuge the bacteria to the bottom, and discard the culture medium;

(c) extracting using the Tiangen Mini Extraction Kit or the Tiangen Endotoxin-Free Mini Extraction Midi Kit;

(d) After the plasmid was extracted, the concentration was quantified using Nanodrop and stored at -20°C.

(3) Plasmid transfer into human cells

(a) Plasmid transfection was performed using the Lipo3000 kit (1.5 μg of plasmid was added to each well);

(b) After transfection, the cells are cultured for 2-3 days to fully perform gene editing and then the cells are recovered;

(c) After the cell culture is completed, the culture medium is aspirated with a pipette tip, and 200 μL of 0.5 M EDTA solution is added to each well of the 12-well plate. After standing for ten minutes, the culture medium is resuspended by pipetting, transferred to an EP tube, and centrifuged at 12,000 rpm for 1 min. The supernatant is taken to recover the cells;

(4) Identification of editing activity

After harvesting the cells, perform genome extraction and T7E1 enzyme digestion assay to detect activity. The steps are as follows:

(a) Genomic DNA extraction: Genomic DNA was extracted using a genomic DNA extraction kit (Tiangen), and the gDNA concentration was measured using Nanodrop;

(b) Targeted region PCR: GXL Prime was used to amplify the target site region from gDNA. The amplification primers are shown in Table 7 below. The deoxynucleotide sequences used were synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform. The PCR purification and gel extraction kit (MN) was used for purification. The cleanliness of the PCR product was analyzed by agarose gel electrophoresis, and the concentration was measured using Nanodrop.

(c) Denaturation and annealing: Use Bio-rad PCR instrument to denature and anneal the purified product of step (c). An equal amount of substrate DNA (about 200-300 ng/rxn, reaction system 10 μl) is added to the T7E1 digestion reaction.

(d) T7E1 digestion: Add 0.2 μl of T7EI nuclease to 10 μl of the sample in step (d) and perform digestion reaction at 37°C for 20 minutes.

(e) Activity detection: After T7E1 completes the cleavage reaction, loading buffer is added and the bands are detected on agarose gel.

Table 7: List of PCR amplification primers

As shown in FIG13 , the sg03 plasmid of BES4 has editing activity in human cells.

In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Appendix I: Novel Type II Crispr-Cas System

Appendix II: New Cpf1 (Type V) System

Claims (10)

1. A Cas protein, comprising:

the amino acid sequence shown in SEQ ID NO. 4.

2. A nucleic acid sequence encoding the Cas protein of claim 1.

3. The nucleic acid sequence of claim 2, wherein the nucleic acid sequence is DNA or RNA.

4. An expression vector comprising the nucleic acid sequence of claim 2 or 3.

5. A recombinant cell comprising the expression vector of claim 4, wherein the recombinant cell is a non-plant cell.

6. The recombinant cell of claim 5, wherein the recombinant cell is a eukaryotic cell.

7. The recombinant cell of claim 6, wherein the recombinant cell is an animal cell.

8. A Crispr-Cas system comprising the Cas protein of claim 1.

9. The system of claim 8, further comprising at least one of: crRNA, tracrRNA or a chimeric RNA formed from crRNA, tracrRNA.

10. Use of the Cas protein of claim 1, the nucleic acid sequence of claim 2 or 3, the expression vector of claim 4, the recombinant cell of any one of claims 5-7, or the Crispr-Cas system of claim 8 or 9 in the field of gene editing for non-disease diagnosis or treatment.