RU2800917C1 - Method of obtaining a knockout of the cd209 gene in bos taurus embryos by transducing zygotes with adeno-associated viruses encoding sacas9 and the corresponding guide rna - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention includes a plasmid obtained on the basis of the px601 vector and encoding the SaCas9 protein and guide RNA against the sequence fragment of exon 3 of the Bos taurus CD209 gene, intended for the assembly of adeno-associated viruses, as well as a method for obtaining Bos taurus embryos and their subsequent transduction with the obtained adeno-associated viruses to obtain mosaic knockouts for the CD209 gene.

EFFECT: development of a method of obtaining a knockout of the CD209 gene in Bos taurus embryos by transducing zygotes with adeno-associated viruses encoding SaCas9 and the corresponding guide RNA.

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Description

Technical field

The invention relates to the field of molecular biotechnology and concerns a method for editing the genome, first of all, obtaining a knockout of selected genes, in this case CD209. The invention can be used to produce animals with a knockout of the CD209 gene, leading to animal resistance to certain pathogens.

The invention was created with the financial support of the Ministry of Science and Higher Education of the Russian Federation under Agreement No. 075-15-2019-1661 dated October 31, 2019 on the basis of the Center for High-Precision Editing and Genetic Technologies for Biomedicine (CVRGTB) of the IBG RAS.

State of the art

Genome editing and modification are modern approaches that make it possible to obtain animals with the required characteristics: milk composition [1, 2], muscle volume [3, 4], or resistance to infectious diseases [5, 6]. To date, genetically modified representatives of various species have been obtained, including rodents [7, 8], primates [9, 10], and cattle [6, 11, 12]. The main method of genetic modification and editing of the animal genome is the introduction of modifications into the genome of embryos at early stages of development. To achieve this goal, various methods are used: transplantation of in vitro genetically modified ESCs into the blastocyst cavity with subsequent formation of a mosaic embryo [13, 14], or delivery of reagents for gene editing to developing embryos by injecting RNA or DNA into the zygote or oocyte [15-20 ] or electroporation of the zygote [21-23]. The main disadvantage of these methods is that they require expensive equipment and highly skilled operators.

Genetic modification of cells (including embryos) by transduction is a simple method: only basic laboratory equipment is required (CO2 incubators, microscopes); no need for microinjections; the viruses used for transduction can be produced elsewhere and are easily transported. Therefore, the production of transgenic embryos can be carried out near farms (where laboratories are often equipped with only basic equipment). Thus, this approach will allow to perform all the necessary procedures (embryo extraction, genetic modification and transfer of embryos to an animal) in one place, reducing the risk of damage to the embryos during transportation.

Various types of viruses can be used to deliver genetic material to the zygote. Retroviruses [24], especially lentiviruses [25], adenoviruses [26], and adeno-associated viruses [27] are used for these procedures.

Adeno-associated viruses (AAV) are widely used in the field of obtaining genetically modified cells and organisms. It has been shown that AAVs serve as an effective tool for obtaining transgenic porcine mesenchymal stem cells [28], spermatozoa and embryos [29], adult mouse neurons [30], mouse [26, 27], and bovine embryos [27].

Adeno-associated viruses have significantly smaller virions compared to retroviruses or adenoviruses. Their small size (about 20 nm) [31, 32] facilitates the penetration of the AAV virion into the embryo.

AAVs have traditionally been considered a safe viral vector platform due to the fact that a helper virus is required for infection to occur in vivo. An adeno-associated virus can infect a cell, but it requires a helper virus, such as adenovirus or herpes simplex virus, for its cycle of reproduction. All serotypes of recombinant AAV vectors generated in the absence of helper viruses are considered risk group 1 agents (agents not associated with disease in healthy adults) unless the transgene encodes for a toxic or potentially tumorigenic gene. Therefore, many recombinant AAV vectors can be used without additional biosafety concerns [33].

Within the scope of this invention, transduction of bovine embryos with AAV has been selected as a potential approach to genome editing. Five different AAV serotypes were used to evaluate their ability to deliver genetic material to bovine embryos.

In mammals, CD209 (also known as DC-SIGN; a dendritic cell-specific cell-cell adhesion molecule) is one of the key proteins involved in many immunological events. CD209 is a C-type lectin receptor present on the surface of macrophages and dendritic cells [34]. Being part of the first line of innate immunity, CD209 recognizes a wide range of pathogens rather non-specifically: viruses, bacteria, fungi, protists. During co-evolution in host/pathogen systems, some pathogens have learned to use CD209 to spread further (for example, CD209+ DCs are used to transport HIV virions from a mucosal contact site to the bloodstream and lymph nodes) and thus infect different cell types. The effect of CD209 on different stages of the infectious process has been shown for HIV [35], hepatitis C virus [36], Ebola virus, Dengue virus, coronaviruses, Mycobacterium tuberculosis [37, 38], Leishmania [39] and other infectious agents (reviewed in [40 ]).

One of the possible mechanisms associated with CD209 to protect pathogens from the immune system has been elucidated for HIV and hepatitis C viruses. It has been shown that even though normally, after binding to CD209, pathogens internalize into proteasomes for future antigen presentation, under certain circumstances (such as the presence additional receptors) after CD209 binding to HCV and HIV, the particles can be internalized into specialized, nonlysosomal, low pH endosomes [36, 41, 42]. It has recently been demonstrated that CD209 knockout mice are less susceptible to Toxoplasma gondii infection: parasite invasion and spread were slower and mortality decreased [43].

It has recently been shown that some isoforms of CD209 can influence the development of resistance of cows to infection with paratuberculosis [44-46]. Moreover, overexpression of CD209 in bovine macrophages in vitro has been reported to lead to increased adenovirus infection [47]. Thus, obtaining a CD209 knockout is relevant for a better understanding of the mechanisms of infection in paratuberculosis and adenoviruses. Obtaining such a knockout could potentially allow animals to become resistant to various infections.

Brief description of the invention

The objective of the claimed invention is to develop a method for obtaining Bos taurus embryos with a knockout of the CD209 gene and obtaining the corresponding embryos.

The problem is solved by using AAVs encoding components of the CRISPR/Cas9 system, an appropriate method has been developed and Bos taurus embryos with a knockout of the CD209 gene have been obtained, for which:

1. Based on the px601 vector (SEQ ID NO: 1), a px601+CD209-sg plasmid construct was obtained containing a guide RNA (SEQ ID NO: 2) against the corresponding region (exon 3 of the Bos taurus gene) of the CD209 gene, under the control of the U6 promoter and ORF of the saCas9 gene under the control of the CAG promoter.

2. A method was developed for obtaining embryos with a null allele of the CD209 gene, characterized by the following stages:

a) Assembly in cells of the HEK293T packaging line of AAV stock containing saCas9 ORF and guide RNA;

b) In vitro fertilization of Bos taurus eggs with Bos taurus spermatozoa;

c) AAV embryo infection;

d) Analysis of the presence of a frameshift in the CD209 gene in blastocysts (d) by amplifying the target region of the CD209 gene with primers cd209-check-f (SEQ ID NO: 3) and cd209-check-r (SEQ ID NO: 4) followed by sequencing of the fragment using primer cd209-check-r (SEQ ID NO: 4).

The technical result of the invention: a method for genetic editing of Bos taurus embryos was developed and embryos with a null allele of the CD209 gene were obtained.

Brief description of the drawings

On FIG. 1 shows a diagram of the mutual arrangement of the first exons of the CD209 gene, guide RNA, and primers for genotype analysis.

On FIG. Figure 2 shows chromatograms obtained as a result of sequencing of mosaic embryos: duplicated sequences corresponding to mosaic embryos in some cells of which genome editing has occurred.

Implementation of the invention

The method is carried out as follows:

1. Selection of guide RNA for making a cut in the bCD209 gene in order to introduce a reading frame shift was carried out using the CRISPOR TEFOR online tool (http://crispor.tefor.net/), taking into account the subsequent use of the SaCas9 nuclease. A guide RNA (CD209-sg, SEQ ID NO: 1) was matched to the third exon of the bCD209 gene. As a result of NHEJ repair after their cutting, a frameshift in the first exon should be formed, leading to a change in the amino acid sequence of the protein and premature formation of a stop codon (Fig. 1). We cloned the selected sgRNA into the px601 plasmid vector designed for simultaneous expression of the saCas9 protein and sgRNA. The guide RNA sequence was synthesized using two oligonucleotides SEQ ID NO: 5, SEQ ID NO: 6.

Vector digestion was performed using BbsI-HF restriction enzyme (New England Biolabs), vector dephosphorylation with FastAP enzyme (Thermofisher Scientific), followed by agarose gel purification and subsequent isolation with the Monarch DNA Gel Extraction Kit.

The oligonucleotides were phosphorylated using the PNK kinase enzyme (Thermofisher Scientific), annealed to each other and ligated to the previously prepared vector using the T4 DNA ligase enzyme (Thermofisher Scientific). The ligation mixture was transformed into competent cells of the XL1blue strain (Evrogen). Colony screening was performed using Isogen PCR kits and primers U6-forv (SEQ ID NO: 7) and the reverse primer used for cloning. Two colonies each containing sgRNA were grown in overnight culture and isolated using the Monarch Plasmid Miniprep Kit. The obtained plasmids were analyzed by Sanger sequencing. The sequencing of the obtained plasmids was carried out using primer U6-forv (SEQ ID NO: 7) by Eurogen. Sequence analysis was performed using Chromas software.

2. The tested construct isolated with the Maxiprep Plasmid Purification Kit (Qiagen) along with Rep2-Cap and pHelper plasmids (both Agilent) was used to transfect HEK293 cells. Recombinant AAVs were obtained as described in [49]. Adherent HEK293T cells were maintained in high glucose DMEM (Gibco, USA) supplemented with 10% v/v. fetal bovine serum (FBS, Biosera Europe, France) and penicillin with streptomycin. Transfection was performed with linear PEI (polyethyleneimine, wt. 25,000) (Polysciences, USA). 72 h after transfection, cells were lysed with 0.5% v/v. Triton X-100, 5 μg/ml RNase A and 10 μg/ml DNase A (Calbiochem, USA) for 1 h at 37°C with shaking. Then, the clarified and concentrated viral suspension was loaded into a modified version of iodixanol (Sigma Aldrich, USA) with a stepwise density gradient (60%, 40%, and 25%) [50] and subjected to ultracentrifugation for 1 h at 350,000 g and 18°C. The fraction containing AAV was dialyzed using a 100 kDa membrane against storage buffer (PBS, 350 mM NaCl, 0.1% Pluronic F-68 (Gibco)), and the purified suspension was further concentrated and sterilized [51]. The titer was determined using a method based on the use of primers and a fluorescent probe targeting ITR (Inverted terminal Repeats) sequences [52]. The absence of protein impurities during preparation was confirmed by silver staining (ThermoFisher, USA).

3. Egg retrieval

Oocytes were isolated from ovaries obtained from local slaughterhouses. The ovaries obtained after slaughter and butchering the carcass were transported to the laboratory within 3-4 hours at a temperature of 38.5 ^° C. The isolation of oocyte-cumulus complexes (containing oocytes and auxiliary cumulus cells) from the ovaries was performed by aspiration as a result of puncturing large follicles. Aspiration was performed using G-MOPS medium (Vitrolife, Sweden) preheated to a temperature of 38.5 ^° C using insulin syringes (1 ml) with a 30G needle.

The resulting oocytes were then subjected to in vitro maturation (IVM - in vitro maturation) to reach the metaphase stage of the second division of meiosis, which is necessary for the possibility of fertilization of oocytes. To do this, oocyte-cumulus complexes were placed in a BO-IVM medium (IVF Bioscience, UK) coated with a layer of paraffin oil (Sage, USA) to prevent evaporation and changes in the osmolarity of the medium, and cultivated at a temperature of 38.5 ^° C in a humidified temperature in an atmosphere of 6.5 % CO2 and 5% O2 in plastic Petri dishes with a diameter of 60 mm.

4. Preparation of spermatozoa

Cryopreserved sperm was used for in vitro fertilization. For cryopreservation, fresh sperm obtained by donation was placed in 0.2 ml wells made in a fluoroplastic plate placed above the surface of liquid nitrogen. The sperm plate was incubated over liquid nitrogen over a height of 5 cm for 1.5-2 minutes, after which it was immersed directly in liquid nitrogen. After removing the liquid nitrogen board, the semen pellets were collected and placed in a Dewar vessel for storage.

Cryopreserved sperm was thawed at 37 ^° C for 1 minute and then centrifuged in a density gradient (80% Percoll SpermGrad (Vitrolife, Sweden)) with a volume of 3 ml for 10 minutes at 700g. After centrifugation, the pellet was washed with SpermRinse buffer medium SpermGrad (Vitrolife, Sweden), supplemented with 5 IU/ml heparin for 5 minutes at 350g.

5. In vitro fertilization

For in vitro fertilization, spermatozoa (at a concentration of ~1*10 ⁶ -2*10 ⁶ per ml) and oocyte-cumulus complexes were placed in BO-IVF medium (IVF Bioscience, UK) for 15 hours for fertilization. The embryos were then washed three times in BO-IVF medium (IVF Bioscience, UK). The procedure of intracorporeal fertilization was carried out in a CO ₂ incubator at a temperature of 38.5°C in a humidified atmosphere containing 6.5% CO ₂ and 5% O ₂ .

6. Embryo transduction and culture

Embryos were cultured in a CO ₂ incubator at 38.5°C in a humidified atmosphere containing 6.5% CO ₂ ; embryos were cultured in BO-IVC medium (IVF Bioscience, UK) coated with a layer of paraffin oil (Sage, USA) to prevent evaporation and changes in the osmolarity of the medium. Embryos were cultured up to the blastocyst stage (170-180 hours of cultivation) without changing the medium.

Embryo transduction was performed using adeno-associated viruses. To do this, AAV virions were added to the medium for cultivating embryos (at the moment when the embryos were at the zygote stage) at a concentration of 5*10 ⁹ viral genomes per 100 μl of the culture medium.

7. Analysis of the genotype of embryos. On day 7 after transduction, the embryos were washed from the medium in TE buffer (10 mM Tris, 1 mM EDTA), lysed in 5 µl of blastocyst lysis buffer (0.1 M Tris-HCl, 0.1 M KCl, 0.02% Gelatin, 0.45% Tween-20, 0.125 mg Proteinase K) for 20 minutes at 56 degrees followed by 10 minutes at 95 degrees. Using the lysate as a template, the target fragment was amplified using 10 pM primers SEQ ID NO: 3 and SEQ ID NO: 4 and Phire Tissue Direct PCR Master Mix (Thermofisher) according to the protocol: denaturation 98°C - 30 sec; followed by 35 cycles: 98°C - 5 sec, 63°C - 5 sec; 72°C - 10 sec; and the last synthesis 72°C - 1 min. After the reaction, the PCR mixture was purified in a 1.5% agarose gel, the resulting product was isolated using the Monarch DNA Gel Extraction Kit, sequenced at Eurogen using primer SEQ ID NO:4. The resulting sequences can be divided into two categories: wild-type sequences, in which the sequence corresponds to the wild-type, and duplicated sequences, demonstrating embryonic mosaicism (Fig. 2).

Example 1. Obtaining Bos taurus embryos with edited CD209 gene in part of the cells. Based on the results of Sanger sequencing, 3 out of 22 tested embryos were found to contain insertions and deletions in the PAM site region. The sequences of these embryos are presented in figure 2. Insertions and deletions occurred at a distance of three nucleotides from the PAM site (the most likely site of cutting SaCas9). The first embryo had a delTTGAACAG deletion and an insAT insertion, the second embryo had a delTTG deletion and an insA insertion, and the third embryo had a delGAACCAG deletion and an insATAG insertion.

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Sequence listing

DTD Version: V1_3

File Name: CD209 in Bos taurus embryos .xml

Software Name: WIPO Sequence

Software Version: 2.1.2

Production Date: 2022-11-03

General Information:

Current application / IP Office: US

Current application / Applicant file reference: none

Applicant name: Federal state budget institution

Sciences Institute of Gene Biology, Russian Academy of Sciences

Applicant name / Language: en

Applicant name / Name Latin: Institute of Gene Biology

Academy of Sciences

Invention title: Method for producing CD209 gene knockout in Bos embryos

taurus by transduction of zygotes with adeno-associated viruses,

encoding saCas9 and the corresponding guide RNA (ru)

Sequence Total Quantity: 7

sequences:

Sequence Number (ID): 1

Length: 7446

Molecule Type: DNA

Features Location/Qualifiers:

-source, 1..7446

> mol_type, other DNA

> organisms, synthetic constructs

Residents:

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cggggcgtcg

ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa

ctccatcact 120

aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact

agttattaat 180

agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc

gttacataac 240

ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg

acgtcaataa 300

tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa

tgggtggagt 360

atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca

agtacgcccc 420

ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac

atgaccttat 480

gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc

atggtgatgc 540

ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacggggga

tttccaagtc 600

tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg

gactttccaa 660

aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta

cggtgggagg 720

tctatataag cagagctctc tggctaacta ccggtgccac catggcccca

aagaagaagc 780

ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa ctacatcctg

ggcctggaca 840

tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac

gtgatcgatg 900

ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg

cggagcaaga 960

gaggcgccag aaggctgaag cggcgggaggc ggcatagaat ccagagagtg

aagaagctgc 1020

tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac

ccctacgagg 1080

ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc

gccctgctgc 1140

acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac

accggcaacg 1200

agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag

aaatacgtgg 1260

ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc

atcaacagat 1320

tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag

aaggcctacc 1380

accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc

cggcggacct 1440

actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa

gaatggtacg 1500

agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg

aagtacgcct 1560

acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc

accagggacg 1620

agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg

ttcaagcaga 1680

agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa

gaggatatta 1740

agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag

gtgtaccacg 1800

acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg

ctggatcaga 1860

ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa

ctgaccaatc 1920

tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag

ggctataccg 1980

gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg

tggcacacca 2040

acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag

gtggacctgt 2100

cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc

cccgtcgtga 2160

agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag

tacggcctgc 2220

ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc

cagaaaatga 2280

tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa

atcatccgga 2340

ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac

gacatgcagg 2400

aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg

aacaacccct 2460

tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac

agcttcaaca 2520

acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc

ccattccagt 2580

acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac

atcctgaatc 2640

tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg

gaagaacggg 2700

acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg

2760

acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac

aacctggacg 2820

tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag

tggaagttta 2880

agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc

attgccaacg 2940

ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg

atggaaaacc 3000

agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag

caggagtaca 3060

aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag

gactacaagt 3120

acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc

ctgtactcca 3180

ccgggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc

ctgtacgaca 3240

aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg

ctgatgtacc 3300

accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac

ggcgacgaga 3360

agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag

tactccaaaa 3420

aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg

aacgcccatc 3480

tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg

tccctgaagc 3540

cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc

gtgaagaatc 3600

tggatgtgat caaaaaagaa aactactacg aagtgaatag caagtgctat

gaggaagcta 3660

agaagctgaa gaagatcagc aaccaggccg agtttatcgc ctccttctac

aacaacgatc 3720

tgatcaagat caacggcgag ctgtatagag tgatcggcgt gaacaacgac

ctgctgaacc 3780

ggatcgaagt gaacatgatc gacatcacct accgcgagta cctggaaaac

atgaacgaca 3840

agaggccccc caggatcatt aagacaatcg cctccaagac ccagagcatt

aagaagtaca 3900

gcacagacat tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct

cagatcatca 3960

aaaagggcaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag

aaaaagggat 4020

cctacccata cgatgttcca gattacgctt acccatacga tgttccagat

tacgcttacc 4080

catacgatgt tccagattac gcttaagaat tcctagagct cgctgatcag

cctcgactgt 4140

gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct

tgaccctgga 4200

aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc

attgtctgag 4260

taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg

aggattggga 4320

agagaatagc aggcatgctg gggaggtacc gagggcctat ttcccatgat

tccttcatat 4380

ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac

tgtaaacaca 4440

aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta

gtttgcagtt 4500

ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa

gtatttcgat 4560

ttcttggctt tatatatctt gtggaaagga cgaaacaccg gagaccacgg

caggtctcag 4620

ttttagtact ctggaaacag aatctactaa aacaaggcaa aatgccgtgt

ttatctcgtc 4680

aacttgttgg cgagattttt gcggccgcag gaacccctag tgatggagtt

ggccactccc 4740

tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg

acgcccgggc 4800

tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg

gcgcctgatg 4860

cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca

aagcaaccat 4920

agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg

cgcagcgtga 4980

ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct

tccttttctcg 5040

ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta

gggttccgat 5100

ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt

tcacgtagtg 5160

ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg

ttctttaata 5220

gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat

tcttttgatt 5280

tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt

taacaaaaat 5340

ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact

ctcagtacaa 5400

tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc

gctgacgcgc 5460

cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc

gtctccggga 5520

gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga

aagggcctcg 5580

tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag

acgtcaggtg 5640

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa

atacattcaa 5700

atatgtatcc gctcatgaga caataaccct gtaaatgct tcaataatat

tgaaaaagga 5760

agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg

gcattttgcc 5820

ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa

gatcagttgg 5880

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt

gagagttttc 5940

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt

ggcgcggtat 6000

tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat

tctcagaatg 6060

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg

acagtaagag 6120

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta

cttctgacaa 6180

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat

catgtaactc 6240

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag

cgtgacacca 6300

cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa

ctacttactc 6360

tagcttcccg gcaacaatta atagactgga tggaggcggga taaagttgca

ggaccacttc 6420

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc

ggtgagcgtg 6480

gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt

atcgtagtta 6540

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc

gctgagatag 6600

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat

atactttaga 6660

ttgatttaaa acttcatttt taatttaaaa ggatctagggt gaagatcctt

tttgataatc 6720

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac

6780

agatcaaagg atcttcttga gatcctttttt ttctgcgcgt aatctgctgc

ttgcaaacaa 6840

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca

actctttttc 6900

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta

gtgtagccgt 6960

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct

ctgctaatcc 7020

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg

gactcaagac 7080

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc

acacagccca 7140

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta

tgagaaagcg 7200

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg

gtcggaacag 7260

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt

cctgtcgggt 7320

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg

cggagcctat 7380

ggaaaaacgc cagcaacgcg gccttttttac ggttcctggc cttttgctgg

ccttttgctc 7440

acatgt

7446

Sequence Number (ID): 2

Length: 26

Molecule Type: RNA

Features Location/Qualifiers:

- source, 1..26

> mol_type, other RNA

> organism, Bos taurus

residences:

gatgctcact caccttgaac cagagt

26

Sequence Number (ID): 3

Length: 22

Molecule Type: DNA

Features Location/Qualifiers:

- source, 1..22

> mol_type, other DNA

> organisms, synthetic constructs

Residents:

tgaacacaag gagccagatg ac

22

Sequence Number (ID): 4

Length: 20

Molecule Type: DNA

Features Location/Qualifiers:

- source, 1..20

> mol_type, other DNA

> organisms, synthetic constructs

Residents:

gcactgaccc tgagccaaat

20

Sequence Number (ID): 5

Length: 25

Molecule Type: DNA

Features Location/Qualifiers:

- source, 1..25

> mol_type, other DNA

> organisms, synthetic constructs

Residents:

caccgcctca aactgtgtag tggtc

25

Sequence Number (ID): 6

Length: 25

Molecule Type: DNA

Features Location/Qualifiers:

- source, 1..25

> mol_type, other DNA

> organisms, synthetic constructs

Residents:

aaacgaccac tacacagttt gaggc

25

Sequence Number (ID): 7

Length: 21

Molecule Type: DNA

Features Location/Qualifiers:

- source, 1..21

> mol_type, other DNA

> organisms, synthetic constructs

Residents:

gagggcctat ttcccatgat t

21

<---

Claims (6)

Fig. 1. Plasmid construct for the assembly of adeno-associated viruses, obtained on the basis of the px601 vector, having the sequence SEQ ID NO: 1, encoding the ORF of the saCas9 gene under the control of the CAG promoter and guide RNA with the sequence SEQ ID NO: 2 against exon 3 of the Bos taurus gene of the CD209 gene under control U6 promoter. 2. A method for obtaining Bos taurus embryos carrying a null allele of the CD209 gene, characterized by the following steps: a) transfection of HEK293 cells, including assembly using the plasmid construct according to claim 1 in cells of the HEK293T packaging line of a stock of adeno-associated viruses encoding saCas9 ORF and guide RNA; b) in vitro fertilization of Bos taurus eggs with Bos taurus spermatozoa; c) infection of embryos with adeno-associated viruses from point (a); d) analyzing the presence of a frameshift in the CD209 gene in blastocysts by amplifying the target region of the CD209 gene with primers cd209-check-f sequence SEQ ID NO: 3 and cd209-check-r sequence SEQ ID NO: 4, followed by sequencing the fragment with using primer cd209-check-r sequence SEQ ID NO: 4.