Name: protocols for crispr/cas9 mutagenesis of the oriental fruit fly bactrocera dorsalis
Uploaded: 2022-09-28T12:40:00
Description: Watch this Scientific Journal Video about Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis at JoVE.com

This work was supported by Shenzhen Science and Technology Program (Grant No. KQTD20180411143628272) and special funds for science technology innovation and industrial development of Shenzhen Dapeng New District (Grant No. PT202101-02).

1. Target design and in vitro synthesis of sgRNA
<ol>
	<li>Predict the structure of target genes of interest and determine the boundaries between exons and introns via bioinformatic analysis of the B. dorsalis genome (software applications used here are listed in the Table of Materials). 
	NOTE: BLAT20 was used to search potential gene loci in the genome. The high-quality RNA-seq reads (transcriptome) were aligned to the acquired...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+toolkit+for+genome+editing+in+a+disease+vector,+Aedes+aegypti:+transgenic+lines+expressing+Cas9+and+single+guide+RNA+induce+efficient+mutagenesis.">Expanding the toolkit for genome editing in a disease vector, Aedes aegypti: transgenic lines expressing Cas9 and single guide RNA induce efficient mutagenesis.</a> The CRISPR Journal. 4 (6), 846-853 (2021).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+in+locusts:+Successful+establishment+of+an+olfactory+deficiency+line+by+targeting+the+mutagenesis+of+an+odorant+receptor+co-receptor+(Orco).">CRISPR/Cas9 in locusts: Successful establishment of an olfactory deficiency line by targeting the mutagenesis of an odorant receptor co-receptor (Orco).</a> Insect Biochemistry and Molecular Biology. 79, 27-35 (2016).</li><li>Liu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+Bombyx+mori+odorant+receptor+co-receptor+(BmOrco)+impairs+olfactory+sensitivity+in+silkworms.">Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms.</a> Insect Biochemistry &amp; Molecular Biology. 86, 58-67 (2017).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+somatic+and+germline+genome+engineering+of+Bactrocera+dorsalis+by+the+CRISPR/Cas9+system.">Efficient somatic and germline genome engineering of Bactrocera dorsalis by the CRISPR/Cas9 system.</a> Pest Management Science. 75 (7), 1921-1932 (2019).</li><li>Bai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+knockout+of+the+eye+pigmentation+gene+white+leads+to+alterations+in+colour+of+head+spots+in+the+oriental+fruit+fly,+Bactrocera+dorsalis.">CRISPR/Cas9-mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis.</a> Insect Molecular Biology. 28 (6), 837-849 (2019).</li><li>Zheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustered+regularly+interspaced+short+palindromic+repeats+(CRISPR)/CRISPR-associated+9-mediated+mutagenesis+of+the+multiple+edematous+wings+gene+induces+muscle+weakness+and+flightlessness+in+Bactrocera+dorsalis+(Diptera:+Tephritidae).">Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9-mediated mutagenesis of the multiple edematous wings gene induces muscle weakness and flightlessness in Bactrocera dorsalis (Diptera: Tephritidae).</a> Insect Molecular Biology. 28 (2), 222-234 (2019).</li><li>Kent, W. 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TransDecoder Available from: <a target="_blank" href="https://github.com/TransDecoder/TransDecoder/releases/tag/TransDecoder-v5.5.0">https://github.com/TransDecoder/TransDecoder/releases/tag/TransDecoder-v5.5.0</a> (2018)</li><li>Robinson, J. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+applications+of+CRISPR-Cas9+for+genome+engineering.">Development and applications of CRISPR-Cas9 for genome engineering.</a> Cell. 157 (6), 1262-1278 (2014).</li><li>Labun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHOPCHOP+v3:+expanding+the+CRISPR+web+toolbox+beyond+genome+editing.">CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing.</a> Nucleic Acids Research. 47, 171-174 (2019).</li><li>Ai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+microinjection+and+knockout+mutant+identification+of+CRISPR/Cas9+genome-edited+Helicoverpa+armigera+(H&#252;bner).">Embryo microinjection and knockout mutant identification of CRISPR/Cas9 genome-edited Helicoverpa armigera (H&#252;bner).</a> Journal of Visualized Experiments. (173), e62068 (2021).</li><li>Xie, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=sgRNAcas9:+a+software+package+for+designing+CRISPR+sgRNA+and+evaluating+potential+off-target+cleavage+sites.">sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites.</a> PloS One. 9 (6), 100448 (2014).</li><li>Gratz, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+of+Drosophila+with+the+CRISPR+RNA-guided+Cas9+nuclease.">Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease.</a> Genetics. 194 (4), 1029-1035 (2013).</li><li>Zhu, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+of+juvenile+hormone+receptor,+Methoprene-tolerant,+induces+black+larval+phenotype+in+the+yellow+fever+mosquito,+Aedes+aegypti.">Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, Aedes aegypti.</a> Proceedings of the National Academy of Sciences. 116 (43), 21501-21507 (2019).</li><li>Laohakieat, K., Isasawin, S., Thanaphum, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transformer-2+and+fruitless+characterisation+with+developmental+expression+profiles+of+sex-determining+genes+in+Bactrocera+dorsalis+and+B.+correcta.">The transformer-2 and fruitless characterisation with developmental expression profiles of sex-determining genes in Bactrocera dorsalis and B. correcta.</a> Scientific Reports. 10 (1), 1-13 (2020).</li><li>Gabrieli, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm-less+males+modulate+female+behaviour+in+Ceratitis+capitata+(Diptera:+Tephritidae).">Sperm-less males modulate female behaviour in Ceratitis capitata (Diptera: Tephritidae).</a> Insect Biochemistry and Molecular Biology. 79, 13-26 (2016).</li></ol>

The CRISPR/Cas9 system is the most widely used gene editing tool and has various applications, such as gene threpy30, crop breeding31, and basic studies of gene fuctions32. This system has already been applied for gene editing in various insect species and has served as an effective tool for functional gene studies in pests. The protocols we present here standardize the procedure of design and synthesis of guide RNAs, collecting embryos, embryo injec...

The Oriental fruit fly, Bactrocera dorsalis, is a cosmopolitan insect pest species that causes damage to over 150 species of fruit crops, including guava, mango, Eugenia spp., Surinam cherry, citrus, loquat, and papaya1. The damage caused in Guangdong Province (China) alone is estimated at over 200 million yuans. Adult females insert their eggs beneath the skin of ripening or ripened fruits, causing decay and abscission of the fruit, which decreases fruit quality and overall yield of the crop2. Since adult fruit flies have great flight capacity and their larvae bore into the fruit skin, insecticides requiring direct contact with the pest perform poorly in the field. Additionally, the extensive use of insecticides has increased the resistance of B. dorsalis against various agricultural chemicals, making control of these damaging pests even more difficult3. Therefore, the development of effective and environmentally friendly pest management strategies is desperately needed.
Recently, with the development of molecular biological tools and high-throughput sequencing technologies, scientists are attempting to develop environmentally-friendly pest management strategies, such as RNAi, that target the functionality of important genes (molecular targets) of various insect pests. Genes that are critical to the survival and reproduction of the pest can be identified through functional gene studies and further serve as potential molecular targets for the improvement of specifically targeted and environmentally friendly pest management tools4. To adapt such strategies to Oriental fruit fly control, effective methods for functional gene research are needed.
The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) endonuclease system was initially discovered in bacteria and archaea and found to be an adaptive mechanism involved in the recognition and degradation of foreign intracellular DNA, such as that introduced by infecting bacteriophages5. In the type II CRISPR system, Cas9 endonuclease is guided by small associated RNAs (crRNA and tracrRNA) to cleave trespassing DNA6,7,8 and has become one of the most widely used tools for gene-editing to date9,10,11,12. Since the CRISPR/Cas9 system has several advantages, such as high efficiency of gene silencing and low cost, it has already been applied for gene editing in various insect species, including Aedes aegypti13,14, Locusta migratoria15, and Bombyx mori16. In B. dorsalis, genes related to body color, wing dimorphism, and sex determination have been successfully knocked out using CRISPR/Cas917,18,19. However, detailed procedures for CRISPR/Cas9 application in this insect remain incomplete. Moreover, some steps provided by researchers for B. dorsalis gene editing are also varied and in need of standardization. For example, the forms of Cas9 were different in published references17,18,19.
This paper provides a systematic method for mutagenesis of B. dorsalis using the CRISPR/Cas9 system, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. This protocol will serve as a useful guide for generating mutant flies for researchers who are interested in the functional gene studies in B. dorsalis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6x DNA Loading Buffer</td>
			<td>TransGen Biotech</td>
			<td>GH101-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial climate chamber</td>
			<td>ShangHai BluePard</td>
			<td>MGC-350P</td>
			<td></td>
		</tr>
		<tr>
			<td>AxyPrep Genomic DNA Mini-Extraction Kit</td>
			<td>Axygen</td>
			<td>AP-MN-MS-GDNA-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>BLAT</td>
			<td>NA</td>
			<td>NA</td>
			<td>For searching potential gene loci in the genome</td>
		</tr>
		<tr>
			<td>Capillary Glass</td>
			<td>WPI&#160;</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf InjectMan 4 micromanipulator</td>
			<td>Eppendorf</td>
			<td>InjectMan 4</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneArt Precision gRNA Synthesis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A29377</td>
			<td></td>
		</tr>
		<tr>
			<td>Hisat2</td>
			<td>NA</td>
			<td>NA</td>
			<td>For aligning the transcriptome to the acquired gene loci</td>
		</tr>
		<tr>
			<td>IGV</td>
			<td>NA</td>
			<td>NA</td>
			<td>For visualizing the results from Transdecoder</td>
		</tr>
		<tr>
			<td>Microgrinder</td>
			<td>NARISHIGE</td>
			<td>EG-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZ2-ILST</td>
			<td></td>
		</tr>
		<tr>
			<td>pEASY-Blunt Cloning Kit</td>
			<td>TransGen Biotech</td>
			<td>CB101-02</td>
			<td>https://www.transgenbiotech.com/data/upload/pdf/CB101_2022-07-14.pdf</td>
		</tr>
		<tr>
			<td>Phenol red solution</td>
			<td>Sigma-Aldrich</td>
			<td>P0290-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette cookbook 2018 P-97 &#38; P-1000 Micropipette Pullers</td>
			<td>Instrument Company&#160;</td>
			<td></td>
			<td>https://www.sutter.com/PDFs/cookbook.pdf</td>
		</tr>
		<tr>
			<td>PrimeSTAR HS (Premix)</td>
			<td>Takara Biomedical Technology</td>
			<td>R040A</td>
			<td></td>
		</tr>
		<tr>
			<td>SAMtools</td>
			<td>NA</td>
			<td>NA</td>
			<td>For generating the sorted bam files</td>
		</tr>
		<tr>
			<td>sgRNAcas9-AI</td>
			<td>NA</td>
			<td>NA</td>
			<td>sgRNA design 
			http://123.57.239.141:8080/home</td>
		</tr>
		<tr>
			<td>Sutter Micropipette Puller Sutter&#160;</td>
			<td>Instrument Company&#160;</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans2K DNA Marker</td>
			<td>TransGen Biotech</td>
			<td>BM101-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Transdecoder</td>
			<td>NA</td>
			<td>NA</td>
			<td>For combining the results of assemble transcripts and gene loci information 
			https://github.com/TransDecoder/TransDecoder/releases/tag/TransDecoder-v5.5.0</td>
		</tr>
		<tr>
			<td>TrueCut Cas9 Protein v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>A36498</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-trace biological detector</td>
			<td>Thermo Fisher Scientific</td>
			<td>Nanodrop 2000C</td>
			<td></td>
		</tr>
	</tbody>
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protocols for crispr/cas9 mutagenesis of the oriental fruit fly bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

This protocol presents detailed steps for the development of B. dorsalis mutants using CRISPR/Cas9 technology, including representative results from gDNA selection, collecting embryos and microinjection, insect maintenance, and mutant screening.
The example of the target site of the selected gene is located in the third exon (Figure 1C). This site is highly conserved, and a single band was detected by gel electrophoresis for the DNA template for synthetic...

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Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...

The oriental fruit fly is a highly invasive and adaptive pest species. Effective methods for functional gene research could help us to develop environmental-friendly pest management strategies. This method is high efficiency and low cost.Our protocol will serve as a useful guide for generating mutant flies for oriental fruit fly researchers. To begin, predict the structure of target genes of interest and determine the boundaries between exons and introns via bioinformatic analysis of the Bactroceras dorsalis genome. Use the commercially available gRNA synthesis kit to generate the designed sgRNA.Resuspend the gRNA product in nuclease-free water. Quantify the concentration using a UV-visible spectrophotometer and store at 80 degrees Celsius prior to use. Place B.dorsalis pupae into a plastic cage, with a rearing condition of 55%relative humidity, 26.5 degrees Celsius, and a 14:10 light/day cycle.When the adults eclose, provide a mixture of sugar and yeast as food along with water. Most adults reach sexual maturity 10 days after emergence. Use a light stand with a light of 30 to 50 lux to improve the fecundity of females, therefore, improving the efficiency of embryo collection.Next, place a 200-mesh gauze in the oviposition chamber, approximately 1 to 2 millimeters away from the chamber lid. Put a new oviposition chamber into the cage when the microinjection setup is ready. Collect the embryos every 10 minutes using a fine wet brush and line them up on a self-made injection plate.Dip the embryos into halocarbon oil during injection to avoid further desiccation. Prepare the working solution by mixing the Cas9 protein and the corresponding sgRNA to a concentration of 300 nanograms per microliters of each. Then, add 1 microliter of phenol red to the mixture to serve as a convenient way to mark injected embryos.Place the prepared mixture on ice to avoid degradation of the sgRNA. Prepare the glass injection needle using a micropipette puller. Add 3 microliters of the mixture into the injection needle without introducing air bubbles.Then, open the needle using a Microgrinder and connect it to the micromanipulator according to the user guide. Set the parameters for the injector as Pi to 500 hectopascals. Ti to 0.5 seconds, and PC to 200 hectopascals.Place the plate with lined-up embryos on the objective table. Then, adjust the micropipette position under a fine optical microscope, setting the micropipette and embryo on the same plane. Insert the needle tip into the posterior, or vegetal pole, of the embryo.Then, press the pedal from the injector to deliver the mixtures into the embryo. The appearance of slight reddish color is due to the addition of phenol red in the mixture. Place the injection plate with the injected embryos into an artificial climate chamber.After 36 hours, transfer the hatched larvae into larval diet using a fine-tip brush. Collect the larvae three or four times daily until no more larvae hatch. Next, place the mature larvae into the wet sand to pupate.The pupation stage takes 10 days. After pupation, move the pupae to a plastic cage before eclosion begins. After isolating genomic DNA from a fresh puparium or a single mid-leg of individual adults and designing the specific primers to amplify the target area, perform polymerase chain reaction, or PCR, following cycling conditions described in the text manuscript.Then, sequence the purified PCR products. Detecting multiple overlapping peaks adjacent to the target site suggests successful mutagenesis. For sub-cloning, mix the purified PCR products with a blunt-end vector and ligate them at 25 degrees Celsius for 15 minutes.Then, add trans-T1 cells and place it on ice for 30 minutes. Then, add 500 microliters of LB medium and incubate at 37 degrees Celsius for 1 hour with shaking. Centrifuge and discard the supernatant.Resuspend the pellet and spread it on an LB plate. Place the plate for overnight incubation at 37 degrees Celsius. Then, select individual bacterial colonies for sequencing to verify the genotype of the mutants.In this study, the example of the target site of the selected gene is located in the third exon. The target site is highly conserved, and a single band was detected by gel electrophoresis for the DNA template for synthetic gRNA and gRNA obtained in vitro transcription. B.dorsalis survival and mutagenesis after microinjections are shown here.After sequencing the PCR products, 80%of the G0 individuals are mosaic mutants. In mutant screening, the mutant with 8-base-pair deletion resulted in premature termination of amino acid translation. Inserting the needle into the posterior area or vegetal pole of an embryo is a central step because this directly affects the survival of eggs.This technological provides a reference for studying areas of gene functions in B.Dorsalis They can quickly capture the mutants they want through this method.

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