Production of varroa destructor parasite resistant jhamt gene silenced genotype in honeybees using crispr-cas9 method

Abstract

The invention relates to male bee sperms in which the target region having the nucleotide sequence SEQ ID NO:1 on the JHAMT (Gene ID: 724216) gene is silenced by the forward primer having the nucleotide sequence SEQ ID NO:2 by means of CRISPR-Cas9 technology, and to the insemination of young queen bees with these sperms via artificial insemination. With the repetition of the same procedures, two generation queen bees were bred, and mutant bees were produced, silencing the Jhamt gene. By means of silencing this gene, bees resistant to Varroa destructor parasite are obtained.

Description

[0001] PRODUCTION OF VARROA DESTRUCTOR PARASITE RESISTANT JHAMT GENE SILENCED GENOTYPE IN HONEYBEES USING CRISPR-CAS9 METHOD

[0002] Technical Field

[0003] The invention relates to the production of a honey bee genotype resistant to the Varroa destructor parasite, which causes irreversible damage to honey bee hives. In the invention, the JHAMT gene is silenced (Knock-out) by applying CRISPR-Cas9 to sperm extracted from adult male bees and young queen bees are fertilised by artificial insemination using these sperms, and honey bee genotypes that the Varroa destructor parasite cannot recognise and therefore cannot harm are obtained.

[0004] State of the Art

[0005] Honeybees (Apis mellifera) are a type of insect that is of great importance to the ecosystem. A honey bee lands on an average of 2000 flowering plants per day for the purpose of collecting pollen throughout its life, and thus enables the pollen of these plants to be transported to different regions, making it possible for the plants to be fertilised. With these journeys they make for the purpose of collecting pollen, honeybees contribute to the reproduction of plants, which are an important part of the ecosystem

[0001] , Experts also consider the existence of honeybees to be of great importance for the preservation of the natural balance of the ecosystem. In addition to these contributions they provide to the ecosystem, honeybees have also found a place in daily human life because of the honey they make. Turkey ranks 3rdin the world with approximately 8 million beehives and 2ndin the world with approximately 120,000 tons of honey production.

[0006] Honeybees live in a colony (hive) that includes a queen bee, female worker bees and male bees. The duty of the queen bee, which is only one in each hive, is to ensure the continuation of the hive through reproduction. The queen's fertilised eggs produce worker bees, and the addled eggs produce male bees. While these female bees that are fed with royal jelly from the larva stage become queen bees, those that are fed with pollen become sterile female bees (worker bees) [2], If the queen dies, a new queen is raised by the rest of the hive; if the queen cannot be raised, deaths begin in the hive.

[0007] Among the reasons that cause death in the hive are the death of the queen bee, as well as damage caused by parasites and harmful chemicals. While diseases caused by parasites are the most common external factors that cause the death of bees, the Varroa destructor parasite is one of the most common causes of death in honey bee colonies in the world [3]. V. destructor enters the hive through the interaction of bees carrying this parasite with healthy bees. Although the bees in the hive try to cope with this parasite, the colonies usually die out. In the event of the spread of the parasite, the strength and immunity of the bees in the hive decreases. This situation causes disruptions in the functioning of the hive. As a result of these disruptions, deaths begin in the hive. As a result of these deaths, great damage occurs in beekeeping. Due to the great damage caused by the . destructor parasite, many precautions are taken to cope with this parasite.

[0008] In the state of the art, the damage caused by the . destructor parasite is tried to be prevented with chemical drugs widely used. Drugs containing active ingredients such as amitraz, thymol, etc. can be effective in killing this parasite [4], However, due to some side effects caused by these drugs, they not only harm honeybees but also indirectly harm ecology and human health. In addition, studies show that these chemicals leave residues in honey.

[0009] RNAi technology, which is in the state of the art, is another method that has been tried to cope with the V.destructor parasite. This method generally involves targeting the vital and reproductive genes of V. destructor with RNAi. As a dsRNA delivery method, dsRNA given to the honey bee through direct transfer, topical application, and feeding is carried to . destructor as an extreme. The most important disadvantages of this method are that it is a supplement to RNAi technology, and the dsRNA stability and the practical effect of the RNAi process are affected. In addition, dsRNA activity must last for a limited time in the hemolymph of the organism and dsRNA must be checked again at the end of this period (72 hours) and these process steps must be carried out in a laboratory environment. On the other hand, there are great concerns that the high homology of the genes targeted in ] / . destructor with genes in honeybees may also cause deaths in honeybees. Therefore, RNAi technology has not gone beyond laboratory studies.

[0010] Due to the limitations and inadequacies of the solutions int eh state of the art, the damage that commonly used chemicals cause to healthy bees, humans, the environment and the honey in the hives, the need to apply RNAi technology in a laboratory environment, the fact that this technology takes a long time and results in death in bees, it has become necessary to develop new methods that are sustainable, environmentally friendly and will give definitive results against Varroa mites.

[0011] Brief Description and Aims of the Invention

[0012] The invention describes a honey bee genotype with the JHAMT gene silenced, making it resistant to the Varroa destructor parasite. This genotype is developed by applying the CRISPR-Cas9 technique to sperm extracted from adult male bees, followed by artificial insemination using the modified sperm.

[0013] The aim of the invention is to produce a honey bee genotype that is resistant to the Varroa destructor parasite. In the invention, the JHAMT gene in these sperms is silenced with CRISPR-Cas9 applied to the sperms of male honeybees and fertilisation is performed with these sperms. Thus, the first generation of bees with the silenced JHAMT gene is obtained. There is no relevant pheromone production in bees with silenced JHAMT gene. By means of the inactivation of the JHAMT gene, which is thought to provide the relationship between Varroa and honeybees, the absence of this pheromone production prevents honeybees from being recognised by the V. destructor parasite. Since the parasite cannot recognise the honey bee, it is not possible for the parasite to meet the bee and thus the bees and the colony are protected from the parasite.

[0014] Another aim of the invention is to quickly provide bee colonies resistant to the V. destructor parasite. In the invention, JHAMT gene silenced homozygous mutant queens can be produced. By means of the production of a queen bee resistant to the . destructor parasite, the colony produced by this queen is also resistant to the Varroa parasite and thus the production of colonies resistant to the . destructor parasite can be carried out very quickly and easily.

[0015] Another aim of the invention is to provide a sustainable and effective solution against the ] / . destructor parasite. By means of the fact that the JHAMT gene silenced bees described in the invention can easily transfer these features to the next generations, the bees are provided with resistance to the parasite on their own without the need for any additional precautions. Since no additional precautions need to be taken against the ] / . destructor parasite, the parasite can be dealt with much more economically.

[0016] Another aim of the invention is to provide a method that does not harm the bees and the environment in combating the V. destructor parasite. Since the JHAMT gene silenced bees described in the invention are healthy bees and none of the structures used during this gene silencing process harm the environment or the bees, the V. destructor parasite can be combated harmlessly.

[0017] Another aim of the invention is to prevent bee deaths. By means of the resistance of the JHAMT gene silenced healthy bees described in the invention to the V. destructor parasite, bee deaths caused by this parasite are prevented.

[0018] Another aim of the invention is to combat the . destructor parasite without the need for a laboratory environment. By means of the rapid colonisation of queen bees from JHAMT gene silenced healthy bees, In many different locations, colonies that are resistant to . destructor parasite can be obtanied by only these queen bees in many locations without needing a laboratory.

[0019] Another aim of the invention is to ensure that CRISPR technology, which is a gene editing technique in combating Varroa, is applied to honeybees. The invention shows that CRISPR technology can be successfully applied to honeybees. Another aim of the invention is to ensure that gene editing is performed in honeybees via sperm using CRIPSR technology. The CRISPR technology mentioned in the invention is applied to the sperm of male honeybees. Thus, gene editing is performed in honeybees.

[0020] Description of Drawings

[0021] Figure 1 : Electrophoresis gel image of sgRNAs synthesised in PCR using sgRNA forward primer for silencing the JHAMT gene

[0022] Figure 2: Agarose gel image of amplicons obtained by PCR amplification of the mutated JHAMT gene region

[0023] Figure 3: Location and design of sgRNA target DNA sequence in the JHAMT gene

[0024] Detailed Description of the Invention

[0025] The present invention relates to the production of bees that became resistant to the . destrcutor parasite by silencing the JHAMT gene by CRISPR-Cas9.

[0026] In the invention, ] / . destrcutor parasite resistant mutant bees are obtained by silencing (knock-out) the Jhamt gene using sgRNA+Cas9 Forward Primer having SEQ ID NO:2 nucleotide sequence designed by target sequence having SEQ ID NO: 1 nucleotide sequence determined in the Jhamt (Gene ID: 724216) gene region on the sperm DNA of male bees and inseminating young queens using these sperm. Forward Primer with SEQ ID NO: 2 nucleotide sequence is used to create a sgRNA pattern synthesised in PCR using sgRNA in vitro Transcription Kit. The first four nucleotides indicate the sequence, the next seventeen nucleotides the T7 polymerase binding site, the next two nucleotides the transcription initiation site, the next 21 nucleotides the target gene site, and the last 14 nucleotides the scaffold template binding site. The reverse primer is designed to bind to the target gene site. Bees resistant to the . destructor parasite, which is the subject of the invention, are produced following the process steps of:

[0027] - Preparation of sgRNA and Cas9 protein,

[0028] - In vitro transcription of sgRNA by PCR,

[0029] - Purification of transcribed sgRNAs,

[0030] - Obtaining sperm from male bees and silencing by transfection with the CRISPR-Cas9 system, transfection of the target sequence with the nucleotide sequence of SEQ ID NO: 1 using the CRISPR-cas9 method with the forward primer having the nucleotide sequence of SEQ ID NO: 2,

[0031] - Artificial insemination of queen bees, and

[0032] - Breeding CRISPR individuals

[0033] Purification of sgRNAs transcribed during the production of bees resistant to the . destructor parasite, which is the subject of the invention, comprises the process steps of:

[0034] - Adding 78 pl of RNase-free water to the reaction mixture to make a total volume of 100 pl and transferring all 100 pl to a 1.5 ml microcentrifuge tube,

[0035] - Adding 30 pl IVT Binding Buffer and vortexing for 5 seconds,

[0036] - Adding 130 pl isopropanol and vortexing for 5 seconds,

[0037] - Placing the IVT RNA Clean-up Spin Column in the collection tube and loading the sample onto the column and centrifuging at 11 ,000 g for 30 seconds at room temperature,

[0038] - Discarding the filtrate and placing the column back into the collection tube,

[0039] - Adding 600 pl IVT Wash Buffer and centrifuging at 11 ,000 g for 30 seconds at room temperature,

[0040] - Discarding the filtrate and placing the column back into the Collection Tube, - Adding 250 pl IVT Wash Buffer and centrifuging at 11 ,000 g for 2 minutes at room temperature,

[0041] - Placing the IVT RNA Clean-up Spin Column in a new 1.5 ml microcentrifuge tube,

[0042] - Adding 10 pl of RNase-free water directly to the silica membrane of the spin column and incubating for 1 minute at room temperature,

[0043] - Centrifuging at 11 ,000 g for 1 minute at room temperature, and

[0044] - measuring the quality and quantity of 1 pl of purified sgRNA with the BioDrop device and detecting 0.9 pg of RNA in 1 pl of sample as a result of the measurement, and storing the sgRNAs at -20°C until used in the transfection process.

[0045] Obtaining sperm used in the production of bees resistant to the V. destructor parasite, which is the subject of the invention, and transfection with the CRISPR-Cas9 system comprises the process steps of:

[0046] - Releasing the endophallus by squeezing the abdomen of 200 mature male bees collected from 10 different hives by hand and collecting the sperm using syringes containing 1 ml of saline (2% gentamicin) solution,

[0047] - Collecting the sperm (~100 pl) in microcentrifuge tubes,

[0048] - Transfecting the sperm cells with sgRNA and Cas9 protein (Ribonucleoprotein (RNP) complex) using Lipofectamine CRISPRMAX Cas9 Transfection Reagent,

[0049] • Preparing a mixture of sperm and CRISPR-lipofectamine complex for artificial insemination,

[0050] • Mixing 100 pl Opti-MEM medium, 10 pl (100 pg) Cas9 protein, 23 pl (~20 pg) gRNA, 250 pl Cas9 Plus reagent in the first tube,

[0051] • Diluting 150 pl CRISPRMAX reagent in 100 pl Opti-MEM medium in the second tube,

[0052] • Incubating the solutions at room temperature for 5 minutes to obtain the Cas9 nuclease / sgRNA / transfectamine reactive complex after transferring them to a separate tube and mixing well, and

[0053] • Silencing the target sequence having the nucleotide sequence SEQ ID NO:1 using the CRISPR-cas9 method with the forward primer having the nucleotide sequence SEQ ID NO:2 by adding the prepared complex to the medium containing sperm cells and incubating at room temperature for 1 hour for transfection.

[0054] In order to obtain JHAMT gene silenced bees, which are the subject of the invention, first sgRNA and Cas9 protein are prepared. Based on the gene sequence for JHAMT (Gene ID: 724216) obtained from the NCBI (National Centre for Biotechnology Information) database, Target Sequence (SEQ ID NO: 1 ) is selected. Then sgRNAs are produced using the Guide-it sgRNA in vitro Transcription Kit with the PCR approach with forward and reverse primers containing the T7 polymerase binding site, sgRNA Target Sequence (SEQ ID NO: 1 ). The Guide-it Scaffold template and reverse primer are provided from the kit components. The produced sgRNAs are evaluated by gel electrophoresis and BioDrop spectrophotometer. Cas9 protein is purchased commercially. PCR components used for amplification of sgRNA template for in vitro transcription of sgRNA by PCR (Table 1.) are combined in a 200 pl PCR tube and briefly vortexed to mix thoroughly and then centrifuged.

[0055] Table 1. PCR components used in sgRNA amplification The reactions are placed in the pre-heated PCR device and the program given in Table 2 below is run.

[0056] Table 2. PCR program required for sgRNA template amplification The resulting 5 pl PCR product is run on a 2% agarose gel with a 50 bp DNA ladder, and the sgRNA templates are seen as a single band of approximately -130 bp. The sgRNA templates are used as templates for the in vitro transcription (IVT) reaction directly without purification. The components given in Table 3 below are combined in a 200 pl PCR tube. After briefly vortexing the reagents, they are centrifuged.

[0057] Table 3. PCR reaction components used for sgRNA IVT

[0058] The reactions are placed in the pre-heated PCR device and the program given in Table 4 below is run. Table 4. PCR program required for sgRNA IVT

[0059] After 4 hours of incubation, 2 pl of Recombinant DNase-1 (RNase-free) is added to 20 pl of IVT reaction. The reagents are briefly vortexed to mix thoroughly and then centrifuged.

[0060] The reactions are placed in the pre-heated PCR device and the program given in Table 5 below is run. The PCR products are then purified using Guide-it IVT RNA Clean-up.

[0061] Table 5. PCR program required for sgRNA incubation

[0062] Before purifying the transcribed sgRNAs, IVT wash buffer is prepared by adding 24 ml of 96-100% ethanol. Then, 78 pl of RNase-free water is added to the reaction mixture to make a total volume of 100 pl. The entire 100 pl is transferred to a 1.5 ml microcentrifuge tube. 30 pl of IVT Binding Buffer is added to this tube, vortexed for 5 seconds, and 130 pl of isopropanol is added and vortexed for another 5 seconds. IVT RNA Clean-up Spin Column is placed in the Collection Tube and the sample is loaded onto the column. The collection tube is centrifuged at 1 1 ,000 g for 30 seconds at room temperature. The filtrate is discarded and the column is placed back into the collection tube. 600 pl of IVT Wash Buffer is added to the collection tube and centrifuged at 1 1 ,000 g for 30 seconds at room temperature. The filtrate is discarded and the column is placed back into the Collection Tube. 250 pl of IVT Wash Buffer is added and centrifuged at 1 1 ,000 g for 2 minutes at room temperature. The IVT RNA Clean-up Spin Column is placed in a new 1.5 ml microcentrifuge tube. 10 pl of RNase-free water is added directly to the silica membrane of the spin column, incubated at room temperature for 1 minute and centrifuged at 1 1 ,000 g for 1 minute at room temperature. Then, the quality and quantity of 1 pl of purified sgRNA is measured with the BioDrop device and 0.9 pg of RNA is detected in 1 pl of sample. sgRNAs are stored at -20 °C until used in the transfection process.

[0063] 10 different male bees are collected from 20 different hives to obtain sufficient sperm. CRISPR-treated queens are kept in several-frame germ hives. In order for gene-edited bees not to pose a risk to the environment, queen bee grids are used in hives to prevent queens and male bees from escaping into nature. At the same time, combs are regularly checked and if male bee combs form, these combs are deformed. The endophallus is squeezed by hand to remove the abdomen of the bees transported to the laboratory in male bee cages and the sperm is collected with syringes containing 1 ml of saline (2% gentamicin) solution. Sperms (~100 pl) obtained from a total of 200 male bees are collected in microcentrifuge tubes. Transfection of sperm cells with sgRNA and Cas9 protein (Ribonucleoprotein (RNP) complex) was performed using Lipofectamine CRISPRMAX Cas9 Transfection Reagent. A mixture of sperm and CRISPR-lipofectamine complex is prepared for artificial insemination. To prepare this mixture, 100 pl Opti-MEM medium, 10 pl (100 pg) Cas9 protein, 23 pl (~20 pg) gRNA, 250 pl Cas9 Plus reagent are mixed in the first tube. 150 pl CRISPRMAX reagent is diluted in 100 pl Opti-MEM medium in the second tube. The solutions are transferred to a separate tube and mixed well, then incubated at room temperature for 5 minutes, thus obtaining the Cas9 nuclease / sgRNA / transfectamine reactive complex. The prepared complex is added to the medium containing sperm cells. Then incubated at room temperature for 1 hour for transfection. The young queens to be artificially inseminated are transported to the laboratory in queen bee cages. Before starting the process, the queens are given CO2 for 2 minutes in styrofoam boxes to make them faint. The queen bee is placed upside down in the tube in the artificial insemination device, with the last 5-6thsegments of her abdomen remaining outside. Here, she is exposed to CO2 application, ensuring that the queen bee remains motionless during the artificial insemination process. The CO2 flow rate was at the level of 35 ml per minute. The queen bee's sting ring is opened under a microscope with the help of ventral and dorsal hooks, the sting ring is first entered with the help of the ventral hook during the opening process, and the last sternum of the ventral is caught and 2 pl of the transfected sperm mixture in the syringe is injected into each queen bee. All procedures are carried out at room temperature (25-28°C). After each insemination process, the abdomen hook, the needle hook with handle, the queen bee holding tube, the queen bee breeding assistant tube and the glass tip at the tip of the syringe are disinfected with 70% ethanol to prevent the queen from being infected. While artificial insemination of each generation is carried out, 3 queen bees are inseminated only with sperm without using the CRISPR solution as a control group. After the insemination process, the queen bees are marked with numbered tags and recorded and each one is transferred to the nucleus hives where there are separate workers. For the breeding of CRISPR individuals, 20 unmated queen bees were artificially inseminated (GO) using the transfected sperm solution. These inseminated queen bees are given to the nucleus hives where there are young workers. The offspring produced by these queen bees theoretically show genetic heterozygous mutant characteristics. Following the egg-laying performance of these queen bees, their larvae are transferred to the queen bee cells and young queen bees are bred. Thirteen of the queen bees produced are inseminated by artificial insemination with sperm taken from 200 males and combined with CRISPR complex (G1 ). The inseminated queen bees are transferred to the nucleus hives where young workers are located and their egg-laying performance is monitored. As a result, worker larvae hatched from inseminated eggs are genetically heterozygous and homozygous mutants (G2). G2 worker bee larvae (G3) are transported to the laboratory to be used in the in vitro Varroa host test. In addition, sample individuals are taken from the generations at each breeding stage and checked for mutants by Sanger sequencing.

[0064] Bio Basic Genomic DNA Extraction Kit is used in total DNA extraction. Quality and quantity controls of the extracted DNA were performed with gel electrophoresis (1% agarose, 1 X Tris-Acetic acid-EDTA (TAE), 1.5 pl Ethidium Bromide) and BioDrop device. For total DNA extraction, honeybee larvae are transferred to pre-numbered 1 .5 ml microcentrifuge tubes and homogenised with sterile plastic rods. A different plastic rod is used for each individual. 300 pl of ACL solution and 20 pl of proteinase K are added to the homogenised tissues. It is incubated for 1 hour in a shaking water bath at 55°C, with occasional vortexing. The samples removed from the incubation are cooled to room temperature, vortexed for 20 seconds, and centrifuged for 5 minutes at 10,000 g. 300 pl of supernatant is transferred to numbered new microcentrifuge tubes and 300 pl of AB solution is added. The tubes are inverted from time to time and kept at room temperature for 2 minutes. The entire mixture is then transferred to the EZ-10 spin column. Centrifuged for 2 minutes at 2,000 g and the filtrate is discarded. 500 pl of washing solution is added, centrifuged for 2 minutes at 8,000 g and the filtrate is discarded. In order to completely get rid of the washing solution, it is centrifuged for an additional 1 minute at 8,000 g. The columns are placed in new 1 .5 ml microcentrifuge tubes. 30-50 pl of elution buffer is added to the middle of the column and incubated for 3 minutes at room temperature. It is centrifuged for 1 minute at 8,000 g to separate the DNA from the column. The obtained DNAs are stored at -20 °C until used in the next step.

[0065] For Genotyping Analysis, PCR is first performed to determine the indels in the JHAMT gene region of CRISPR individuals. For this purpose, primers (Forward Primer (SEQ ID NO: 2) and Reverse Primer (SEQ ID NO: 3) specifically designed for the target gene on DNA and given in Table 6, reaction components given in Table 7 and PCR program given in Table 8 are used. The nucleotide sequences of JHAMT gene fragments amplified by PCR are determined bidirectionally by Sanger sequencing method. Thus, the distinction between individuals with and without mutation becomes definite.

[0066] Table 6. Descriptive information of primers designed specifically for the JHAMT gene for PCR.

[0067] Table 7. PCR reaction components required for amplification of the JHAMT gene

[0068] Table 8. PCR program required for amplification of the JHAMT gene PCR products are checked by running 1 .5 pl Ethidium Bromide containing 2% agarose gel at 80 volts for 40 minutes. 1 X TAE buffer is used as a buffer solution in electrophoresis.

[0069] Varroa samples are obtained from male bee combs in colonies for the Varroa host preference test. The sections in the combs brought to the laboratory environment are opened with the help of forceps and Varroa are collected one by one and fed with male bee pupae in the white-eye phase in petri dishes until they are used in the test. Varroa are kept in petri dishes in an incubator set at 28° C and 75% relative humidity (RH). To determine the role of the JHAMT gene in Varroa host preference, 2 worker larvae (4thinstar stage) are placed in a 100 x 20 mm petri dish at equal distances (4 cm) from the centre. One of the larvae is selected from the knockout mutant and the other from the control group worker larvae. A single adult female Varroa is placed in the exact centre of the box using a brush. A stopwatch is set from the moment the Varroa is placed and 1 hour is waited. During this period, each mite is checked every 15 minutes and its location is noted. The boxes are covered with a lightproof cover and kept in a room at 34.5°C and 60-70% relative humidity. At the end of the test, Varroa that prefer mutant larvae are scored as “preference (1 )”, Varroa that prefer control larvae are scored as “no preference (0)” and mites that do not show any preference are scored as “not evaluated (?)”. Each mite and petri dish are used only once. A total of 30 tests are performed to obtain sufficient sample size. In addition to this test, a total of 6 comb samples, one from the knockout colonies and one from the control group colonies, are taken, and 100 randomly selected cells from each comb in the laboratory are examined for the presence of mite-infested larvae and pupae. In this way, the effect of the JHAMT gene on the preference status of Varroa in vivo conditions is also determined. The genotyping analysis findings are then examined. While the gene-edited workers are being bred, the JHAMT gene region is amplified by PCR and the DNA sequence is determined by the Sanger sequencing method in order to verify whether the individuals obtained in each generation are mutant. The queens and generations used in the study are created. 20 queens inseminated with transfected sperm are numbered and given to the nucleus hives (GO). Of these queens, 7 (638, 828, 945, 975, 989, 1045, Z) are excluded from the experiment due to failure to lay eggs, refusal of worker bees in the nucleus hive, or other biological reasons. The experiment is carried out with 13 queens (GO) that were inseminated with transfected sperm and started laying eggs healthily within 5 days. 13 young queens thought to be heterozygous mutants are bred from these 13 queens by larva transfer method (G1 ). At the same time, a sample is taken from the worker bee larvae (G1 larva) of GO queens, DNA is isolated, and the JHAMT gene region containing the sgRNA target is amplified by PCR. Sanger sequence analysis of the amplicons obtained as a result of PCR is performed. According to the sequencing results, it was determined that the targeted mutations in the sgRNA target region of 6 out of 13 mothers artificially inseminated with CRISPR-Cas9 transfected sperm in the G1 generation were successfully achieved. CRISPR-Cas9 applications are repeated to breed worker bees (G2) with silenced JHAMT genes from G1 queens. The egg-laying period of inseminated queen bees does not exceed 6 days. A larva (G2) sample is taken from 8 healthy G1 queens that were artificially inseminated with transfected sperm and DNA is isolated, and the JHAMT gene region containing the sgRNA target is amplified by PCR. Sequence analysis of the amplicons obtained as a result of PCR is performed. According to the sequence results, it is determined that mutations were fully achieved in the sgRNA target region in 3 larvae out of the G2 larvae produced by 8 G1 queens. When the JHAMT gene DNA sequence of the G2 control group is examined in the DECODR software, it is determined that there is no mutation in the control group individuals. When Jhamt Genei sequence of G2 larvae of one of the G1 queens is examined in the DECODR software, it is determined that the CRISPR -CAS9 application was successful and the gene regulation in the larvae of this queen was determined by 60.6 %-44, -106 deletion / frame shift and 39.4 %-45 deletions occurred. When Jhamt gene sequence of the G2 Larva of another G1 Queen is examined in the DECODR software, it is determined that the CRISPR -CAS9 application is successful and the gene regulation in the larvae of this queen was determined by 80.1 %of -45 %of -45 %, 19.9 %-46 deletion / frame shift. When Jhamt gene sequence of the G2 Larva of G1 Queen is examined in the DECODR software, it is determined that the CRISPR -CAS9 application is successful and the gene regulation in the larvae of this queen is realized and 100 %of -8 deletion / frame shifts occur at a rate of 100 %. In order for the Jhamt gene to be knocked out, the indeli in the larvae must result in the mutations of the frame shift. Therefore, the indel shapes that occur in individuals are examined separately. In the results of we obtained, 2 of the 3 hives show heterozygous and 1 homozygous frame shift mutation. In the results of the in vitro host test applied for Varroa's host preference test findings, the result of each test is noted according to Varroa's host preference. In 21 (70%) of the 30 tests, the mites prefer control group larvae. It is thought that control larvae and mutant larvae are tested in the same petri container and the ferments emitted by the control larvae spread to the petri container and therefore 9 Varroa is directed to the surprise mutant larvae. As a result of the Ki-square test to determine whether the silence of the Jhamt gene affects Varroa's host preference, it is determined that the silence of the Jhamt gene is effective in Varroa's host preference (p = 0.002).

[0070] When a total of 6 honeycomb samples from the control group and the three pieces taken from the knock out mutant colonies are examined, varroa cannot be detected in any of the gene arranged honeycombs. Varroa is detected in 25 %of the sections opened in the control group honeycomb. Mann Whitney U test is performed to determine the effect of the Jhamt gene's effect on the honeycomb section preference of Varroa. In the results obtained, the Jhamt gene is effective in the choice of honeycomb in vivo conditions of Varroa (p = 0.037 <0.05). REFERENCES

[0071] [1] Pate, V., Pauli, N., Biggs, E., Barbour, L., Boruff, B. “Why bees are critical for achievning sustaniable development”. Ambio. (2021 Jan); 50(1): 49-59

[0072] [2] Akyol, E. “Bal Anlannda Yumurtanm Yapisi ve Embriyo Geli§imi”. U. An Drg., Kasim 2007

[0073] [3] Akyol, E., Korkmaz, A. “Bal Ansi (Apis melliera) Zararlisi Varroa destructor’un

[0074] Biyolojisi”. Uludag Ancihk Dergisi, Agustos 2005-5

[0075] [4] Tutkun, E. “An Akan (Varroa Destructor) Mucadelesinde Timol’un Kullamlmasi”.

[0076] Ancihk Ara§tirma Dergisi, Cilt:8, Sayi:1 , 2016, Sayfa:1 -5

Claims

CLAIMS1. ] / . destructor parasite resistant bee genotype production method, comprising the process steps of silencing the Jhamt gene using sgRNA+Cas9 Forward Primer having SEQ ID NO:2 nucleotide sequence designed by target sequence having SEQ ID NO: 1 nucleotide sequence determined in the Jhamt (Gene ID: 724216) gene region on the sperm DNA of male bees and inseminating young queens using these sperm, thereby obtaining ] / . destrcutor parasite resistant mutant bees.

2. A method according to Claim 1 , wherein the forward primer having the nucleotide sequence SEQ ID NO:2 consists of 58 bases.

3. A nucleotide sequence according to Claim 2, wherein it consists of 58 bases.

4. A nucleotide sequence according to Claim 3, wherein the first 4 nucleotides are the extra sequence, the next 17 nucleotides the T7 polymerase binding zones, the next 2 nucleotides the transcription starting zones, the next 21 nucleotides the target gene region, and the last 14 nucleotidines the Scaffold template binding zone.

5. ] / . destructor parasite resistant bee genotype production method, comprising the process steps of:- Preparation of sgRNA and Cas9 protein,- In vitro transcription of sgRNA by PCR,- Purification of transcribed sgRNAs,- Obtaining sperm from male bees and silencing by transfection with the CRISPR-Cas9 system, transfection of the target sequence with the nucleotide sequence of SEQ ID NO: 1 using the CRISPR-cas9 method with the forward primer having the nucleotide sequence of SEQ ID NO: 2,- Artificial insemination of queen bees, and- Breeding mutant (knock-out) individuals in terms of JHAMT gene.

6. A method according to claim 4, wherein the purification of transcribed SGRNAs comprises the steps of:- Adding 78 pl of RNase-free water to the reaction mixture to make a total volume of 100 pl and transferring all 100 pl to a 1.5 ml microcentrifuge tube,- Adding 30 pl IVT Binding Buffer and vortexing for 5 seconds,- Adding 130 pl isopropanol and vortexing for 5 seconds,- Placing the IVT RNA Clean-up Spin Column in the collection tube and loading the sample onto the column and centrifuging at 11 ,000 g for 30 seconds at room temperature,- Discarding the filtrate and placing the column back into the collection tube,- Adding 600 pl IVT Wash Buffer and centrifuging at 11 ,000 g for 30 seconds at room temperature,- Discarding the filtrate and placing the column back into the Collection Tube,- Adding 250 pl IVT Wash Buffer and centrifuging at 11 ,000 g for 2 minutes at room temperature,- Placing the IVT RNA Clean-up Spin Column in a new 1.5 ml microcentrifuge tube,- Adding 10 pl of RNase-free water directly to the silica membrane of the spin column and incubating for 1 minute at room temperature,- Centrifuging at 11 ,000 g for 1 minute at room temperature, and- measuring the quality and quantity of 1 pl of purified sgRNA with the BioDrop device and detecting 0.9 pg of RNA in 1 pl of sample as a result of the measurement, and storing the sgRNAs at -20°C until used in the transfection process.

7. A method according to claim 4, wherein obtaining the sperms and transfering these with CRISPR-CAS9 system comprising the process steps of:- Releasing the endophallus by squeezing the abdomen of 200 mature male bees collected from 10 different hives by hand and collecting the sperm using syringes containing 1 ml of saline (2% gentamicin) solution,- Collecting the sperm (~100 pl) in microcentrifuge tubes,- Transfecting the sperm cells with sgRNA and Cas9 protein (Ribonucleoprotein (RNP) complex) using Lipofectamine CRISPRMAX Cas9 Transfection Reagent,• Preparing a mixture of sperm and CRISPR-lipofectamine complex for artificial insemination,• Mixing 100 pl Opti-MEM medium, 10 pl (100 pg) Cas9 protein, 23 pl (~20 pg) gRNA, 250 pl Cas9 Plus reagent in the first tube,• Diluting 150 pl CRISPRMAX reagent in 100 pl Opti-MEM medium in the second tube,• Incubating the solutions at room temperature for 5 minutes to obtain the Cas9 nuclease / sgRNA / transfectamine reactive complex after transferring them to a separate tube and mixing well, and• Silencing the target sequence having the nucleotide sequence SEQ ID NO:1 using the CRISPR-cas9 method with the forward primer having the nucleotide sequence SEQ ID NO:2 by adding the prepared complex to the medium containing sperm cells and incubating at room temperature for 1 hour for transfection.