4. Discussion

2.8. Bioassay

To assess the resistance level of mutants, drones were caught a day before the topical application and allowed to feed comb honey ad libitum. On the day of bioassay, ages of each drone ranged from 2 to 6 day after emergence. Although all drones did not qualify as good for bioassay, any drones that were able to walk longer than 5cm were used. A total of eight drones from the OF12 queen and 12 drones from the OF18 queen were used for bioassay.

Analytical standard grade Spinosad (Merck KGaA, Darmstadt, Germany) was diluted with acetone to 100mg/ml, and 1 ml aliquot was topically applicated to the notum of each drone (100mg spinosad/drone). The time until death of each drone was recorded, and drones were then sampled for sequencing.

2.9. Sequencing of F1 drones

A total of 68 drones in various stages were sampled for gDNA extraction and PCR. The samples included dead larvae, pupae, and adults, along with those collected right after bioassay. Parts of collected specimens (2 to 3 legs of adults or equivalent volume of larval/pupal tissue) were flash-frozen with liquid nitrogen in a 1.5-ml centrifuge tube, pulverized with chilled pestle, and then used for gDNA extraction using DNeasy Blood and Tissue kit (QIAGEN). The 316-bp gDNA

of 62 samples passed quality check by gel electrophoresis and their nucleotide sequences were analyzed by the Sanger sequencing method.

3. Results

3.1. Fluorescent imaging of early embryos.

Florescent images of honey bee embryos in different cleavage cycles were obtained to confirm optimal injection site depending on the age of embryos.

Unfortunately, I had difficulties in obtaining a large number of high-quality samples mainly due to the delicacy and opaqueness of honey bee embryos, which only allowed rough observation of the process. Nevertheless, my observation conformed to the previous observations by Schnetter et al.(Fleig & Sander, 1986;

Schnetter, 1934; Yu & Omholt, 1999)

At 1-2 h AEL, nuclei were found in the anterior end of embryos. As presented in the figure 3a, maternal nuclei at its end of meiosis were seen, whereas a single male nucleus were observed in the middle of anterior cytoplasm. The embryo fixed at 3-4 h AEL had four nuclei, implying the stage to be in-between the 2^nd and 3^rd mitotic cycles (Fig 3b). Sixteen pairs of nuclei, which refer to the end of the 4^thmitotic cycle, were observed in the endoplasm of 4-5 h embryo (Fig 3c). At 6-7 h AEL, approximately 60 to 200 nuclei were observed (Fig 3d, e), whereas roughly 500 nuclei were observed in the embryo fixed 8-9 h AEL (Fig 3f). This information was reflected in establishing the injection scheme for more reasonable

Figure 3. Fluorescent image of honey bee embryos in early developmental process. The cleavage cycle over time can be estimated from the number of nucleus.

3.2. in-vitro cleavage assay

Most of the substrate DNA was cleaved by the sgRNA-Cas9 RNP, thereby indicating robust biding activity of both synthesized sgRNA and Hifi Cas9 enzyme. The RNP retained activity after more than five freeze-thawing. Since the RNP showed in-vitro activity in injection buffer, the same RNP solution was directly employed to microinjection.

Figure 4. Digestion of nAChR α6 PCR product by sgRNA-Cas9 RNP. The 615 bp PCR product was digested into 459 and 156 bp fragments.

3.3. Microinjection, grafting, and maintenance

A total of 1667 eggs were injected with various injection conditions (table 2). A total of 253 larvae (15.2%) hatched from the injected eggs and moved to plastic dish prepared for in vitro rearing. Next, I selected 85 larvae for grafting based on healthy appearance and representativeness of each injection batches. A total of 15 queens (17.6%) emerged from the queen cell, and each queen was introduced to exclusive nucleus hives. I lost 12 queens during introduction and maturation process of virgin queens, leaving only three queens that successfully laid unfertilized eggs, which developed into drones. These queens were named after the injection batches, OF12, OF13, and OF18, respectively. Queens began to lay eggs more than a month after emergence.

Although the hatching rate varied even between the batches that were injected under similar conditions, overall tendency of increasing hatching rate depending on the larval age was observed (Fig 5).

Table 2. Injection conditions and results from following procedures. The number of hatching embryos, grafted embryos, emerged queens and queens with offspring is also noted in the table.

Figure 5. Plot of injection batches based on average age of embryos at injection and hatching rate. Hatching rate of embryos increased with the embryonic age at injection.

3.4. Sequencing

The OF12 queen, which was injected at 6.4 h on posterior end, produced 29 offsprings, one (3.4%) of which was determined to be a mutant. The OF13 queen, which was injected at 6.9 h to medial point, produced 10 offsprings, with none of which being mutant. OF18 queen, which was injected at 4 h to medial point, produced 23 offsprings, with 7 (30.4%) of which being mutants (Table 3).

A total of eight mutant drones, displaying seven different genotypes, were detected from sequencing of 63 drones. Six drones, including four adults, had

gene (Fig 6). Any knock-in event was not detected.

Table 3. Injection condition of the queens and sequencing result of each queen.

Two out of three queens (66.7%) had genome edited offsprings.

3.5. Bioassay and phenotype observation.

Survival time for each drone was recorded after topical application of spinosad, with the shortest time being 3 h whereas the longest time marked 24.5 h. Among 20 drones tested, only two drones were confirmed as mutant in the subsequent sequence analysis. Mutants drones showed survival times that are similar to those of the wildtype group (fig 7) and did not display any noticeable difference in appearance compared to the wildtype drones (fig 8).

Figure 6. Indels detected from the mutant F1 drones. 7 different types of indels were found from 8 mutant drones.

First two digits of the drone’s name indicate the name of the queen which laid the drones. Drone 18-8 and 18-18

Figure 7. Bioassay result of F1 Drones. The two KO mutant died at 7 and 14 h after topical application of spinosad, both of which belongs to the range recorded in wildtype drones.

Figure 8. Dorsal (a) and ventral (b) view of nAChR α6 KO Drone (18-8).

4. Discussion

In this study, I examined the potential of genome editing technique in generating a honey bee breed with insecticide resistant traits. In order to prove my concept, I selected the honey bee nAChR α6 as the target of editing and demonstrated that honey bee nAChR α6 gene can be knocked-out by injecting Cas9-sgRNA RNP, and adult drone can emerge from haploid egg even without fully functional nAChR α6. To the best of my knowledge, this is the third successful genome editing following Kohno et al. (Hiroki Kohno & Kubo, 2018;

H. Kohno et al., 2016) and fifth if transgenic honey bees are included (Otte et al., 2018; Schulte et al., 2014).

Also, I showed that FECS can be readily applied to honey bee genome editing.

Plentiful supply of honey bee embryos allowed microinjection with various conditions as well as the unprecedented number total injection counts, both of which can facilitate the optimization of honey bee genome editing technique.

Despite the limited information on honey bee germline formation, honey bee germ cells are also assumed to originate from nuclei in the posterior end of embryo as in most other insects. Based on this assumption, I planned an injection scheme with different injection time and spot from those of Kohno et al. and

Schulte et al. (Hiroki Kohno & Kubo, 2018; H. Kohno et al., 2016; Schulte et al., 2014), both of which injected to posterior end of embryos within 3 h AEL.

Figure 9. Genome editing scenario expected in embryos injected with different conditions. White circles indicate the location where germline is assumed to develop. Orange dots indicates genome edited nuclei, whereas blue dots indicate nuclei which were not affected by Cas9 RNP. OF18, in accordance with the scenario, showed the highest germline genome editing rate.

Although it is almost impossible to draw meaningful conclusion from the limited number of observations, which is only one sample per condition, the

9). The OF18 queen, which was injected to medial region at 4 h AEL, showed a high germline genome editing rate of 30.2%. In contrast, when injected either at 6.9 h to medial region (OF13) or 6.4 h to posterior end (OF12), the germline genome editing rates were reduced to 0% or 3.4%, respectively. It appears that the injection to the medial region of embryo at a relatively early embryonic stage when smaller numbers of nuclei exist (i.e., 4 h AEL) increased the ratio of genome-edited cells compared to the injection at a later time (i.e., 6.4 AEL). Thus, it can be speculated that the injection timing (i.e., age of embryo at injection) is one of the factors determining the success rate of germline genome editing. The high germline genome editing rate of OF18 which targeted medial part of embryo at 4 h AEL, also supports the result of Otte et al.(Otte et al., 2018), which achieved increased germline transformation rate by adopting anterior injection to early stage of embryo (i.e., 1.5 AEL). Further optimization can be achieved when more samples with different injection timing and spot are available, and the genome editing rate in the ovary is analyzed through deep sequencing.

One of the problems during this study was the low hatching rate of injected embryos. Although different injection batches are expected to produce inconsistent results, those with similar conditions (e.g. OF15 to 16 and OF14 to 28) also showed fluctuating results, implying that other factors, along with expertise

exists room for further optimization of protocol. One of the suspicious factors is the formation of dewdrops, which is attributed to high humidity in the storage condition and temperature gap between eggs and storage condition. As the temperature of the room with the incubator was maintained at room temperature (i.e. 24˚C), it is possible that the temperature of the embryo went down to certain point which can induce the formation of dewdrops when the storing process was delayed by any reasons. Success rate of grafting, which was another bottleneck in this experiment, would be increased by grafting into multiple queenless colonies with a large worker population or by imitating commercial royal jelly production in the field that exploits strong queenright colonies and intensive feeding.

Although regulation problem may arise, there would be a low chance of mosaic queen to be released to environment if the queen cells are harvested several days before emergence. The problem of low acceptance rate of virgin queen can be solved by introducing queen cells into nucleus hives as described by Kohno and Kubo (Hiroki Kohno & Kubo, 2018).

In my experiment, mutant drones with frameshifting emerged as adult drones, which indicates the dispensability of the nAChR a6 gene during embryonic, larval, and pupal development. However, it is not certain how much fitness cost will be imposed to α6 KO mutant honey bee. Examining behavioral changes in α6

assessment of the commercial use of spinosad-resistant honey bee, adopting mutants with point mutation which preserve the gene’s function will be forced to minimize the selection pressure and any other types of potential effect that can arise from the KO event. Knock-in (KI) event through HDR is known to occur in a lower likelihood (~10%) compared to KO event through NHEJ (Chu et al., 2015;

Maruyama et al., 2015). Considering this factor and the small (i.e. eight) number of KO mutants identified so far, KI mutant is likely to be obtained if an additional screening with large numbers of drones is conducted. It will be reasonable to keep screening until more than 30 KO mutants are found.

Bioassay of mutant drone was difficult due to the limited number of drones available as well as the suboptimal physical condition of the drones. Drones (both mutants and wild types) grew up in the worker cells during late autumn, being cared by limited number of workers (less than 200 / nucleus hive). For this reason, more than 1/3 of the drones died within 5 days after emergence, and the physical condition of surviving drones were also noticeably bad, making the bioassay more unreliable. Precise bioassay will be conducted when enough number of mutants can be supplied during spring season. Since the spinosad resistance through KO events was detected from three other order of insects (Baxter et al., 2010; Wan et al., 2018; Zimmer et al., 2016), nAChR α6 KO honey bees also is likely to display

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KOREAN ABSTRACT

실리콘 코팅을 적용하여 필름의 부착력을 높이고 손쉬운 탈부착이 가능하도록 하였다.

FECS 를 통한 꿀벌 알의 대량수집을 통해 CRISPR-Cas9 기술을 이용한 꿀벌 유전체 편집을 진행하여 니코틴성 아세틸콜린수용체 알파 6 서브유닛 유전자가 편집된 꿀벌을 개발하는데 성공하였다. 기존 연구에서는 Cas9 mRNA 를 이용하여 진행되었으므로, 이는 Cas9 단백질 주입을 통한 꿀벌 유전체 편집으로는 최초의 보고이다. 비록 형질전환 개체수의 부족으로 정상적인 생물검정이 진행되지는 못하였으나, 알파 6 유전자가 낙아웃 된 꿀벌이 성충 수벌로 정상적으로 발생할 수 있음을 입증하였으며, 후속연구를 통해 스피노사드 저항성 및 기타 표현형을 분석할 수 있을 것으로 기대된다.

검색어: 꿀벌, Apis mellifera, 알, 배아, 마이크로인젝션, 크리스퍼,

유전체 편집, 낙아웃, 스피노사드, 저항성, 니코틴성 아세틸콜린 수용체, 알파 6

,

학번: 2016-21740