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.
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.
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.
1. Target design and in vitro synthesis of sgRNA
2. Embryo collection and preparation
3. Microinjection of the embryo
4. Post-injection insect rearing
5. Mutant screening
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 gRNA (Figure 1D) and gRNA obtained by in vitro transcription (Figure 1E).
Injection into 200 freshly harvested eggs was performed as described in section 3. The embryos were maintained by following the protocols described in steps 4.1-4.3. As detected by sequencing the PCR products, 80% of the G0 individuals are mosaic mutants (Table 1). Here, mutants with 8 bp deletion which resulted in premature termination of amino acid translation in G1 were selected (Figure 3C). This should lead to changes in the corresponding functions of this gene product in B. dorsalis. The selected G1 were crossed with wild-type to obtain G2 heterozygotes. Self-cross the G2 heterozygotes and homozygotes were recovered in the next generation, demonstrating that this scheme is successful for developing B. dorsalis mutants and could be more widely applied in functional gene studies in this and closely related species.
Figure 1: Preparation of sgRNA. (A) General procedures for sgRNA preparation. (B) Target area amplified by PCR from gDNA (100 bp). (C) Example of target gene structure and target cleavage site by Cas9. (D) PCR assembly of sgRNA (~100 bp). (E) Synthesis of sgRNA by in vitro transcription and purification. Please click here to view a larger version of this figure.
Figure 2: Embryo microinjection. (A) The process and method of injection. (B) Embryo collection device, laying eggs on gauze. (C) Line up embryos with water and cover with halocarbon oil. (D) Micropipette puller. (E) The whole setup of the microinjection system. Microscope (left), microinjector and micromanipulator (middle), and automatic air pump (right). Please click here to view a larger version of this figure.
Figure 3: Mutant Screening. (A) Mating of mutants and acquisition of homozygotes. (B) PCR products from the mutant genome which generates a distinct set of peaks at the target. (C) One of the mutation types and amino acid changes. Deletion mutations lead to premature termination of translation. Abbreviations: HE = heterozygotes, HO = homozygotes. Control: embryo injected with scrambled sgRNAs. Please click here to view a larger version of this figure.
Injection mix | Injected embryos | Hatched larvae | Pupae | Mosaic G0 | ||
(Mosaic/Total adults) | ||||||
BdorOrco_gRNA1 (300 ng/μL) | 200 | 83 | 66 | 49/60 (81%) | ||
BdorOrco_gRNA2 (300 ng/μL) | ||||||
Cas9 (150 ng/μL) | ||||||
Phenol red (1 μL) | ||||||
BdorOrco_scrambled gRNA1 (300 ng/μL) | 200 | 78 | 76 | 0/70 (0%) | ||
BdorOrco_scrambled gRNA2 (300 ng/μL) | ||||||
Cas9 (150 ng/μL) | ||||||
Phenol red (1 μL) |
Table 1: B. dorsalis survival and mutagenesis after microinjections.
Problem | Potential cause(s) | Solutions | |
Inefficient embryo collection | 1. Adults are in poor condition | 1.Ensure the availability of food and water, especially the sugar content of the food. | |
2. Insufficient mating | 2. Mating in advance under 30-50 Lux dim conditions. It is best to select 12-15 days-old mated adults. | ||
Poor hatchability of eggs after injection | 1. Poor quality embryos | 1. Pick fresh and plump eggs and gently brush them with a water-soaked brush. | |
2. Needle does not fit | 2. The tip of the needle is as small as possible to minimize damage to the egg during injection. | ||
3. Improper rearing of eggs after injection | 3. Eggs need to be kept hydrated after injection to prevent dehydration from drying out. Eggs can also be transferred directly to food after injection. | ||
Low larval survival rate | 1. Moldy food is not suitable for larvae growth | 1. For newly hatched larvae, add a small amount of food. Too much food will mold or dry out and affect the growth of the larvae. | |
2. Newly hatched larvae soaked in oil for a long time | 2.The newly hatched larvae need to be transferred to the larval food in time or the eggs should be picked directly into the food after injection. | ||
Adult mutation rate is low | 1. Low quality of gRNA | 1. Rigorous experimental operation to synthesize high-quality gRNA to prevent degradation. | |
2. Inefficient targets lead to off-targets | 2. Screening high-efficiency targets through as our discussion mentioned. |
Table 2: Possible problems with constructing mutants, potential causes, and solutions.
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 injection, insect rearing, and mutant screening. Moreover, a troubleshooting table was added to summarize the potential problem from each step, and their solutions were provided to lighten the workload and improve efficiency (Table 2). This procedure provides a reliable way to edit genes of interest and improves the functional genomic studies in B. dorsalis.
First, target gene selection is critical to increasing the efficiency of the CRISPR/Cas9 system, and several open source software programs such as ChopChop33,34 (http://chopchop.cbu.uib.no/), sgRNAcas9 (V3.0)35, and CRISPR optimal target finder36 (http://targetfinder.flycrispr.neuro.brown.edu) can automatically generate targets and predict potential off-target rates. Since the high fecundity rate of B. dorsalis facilitates embryo collection, mutant rates can be predicted by sacrificing a portion of the embryos for DNA extraction and sequencing of the target region. If sequencing is not available in the lab, using T7 endonuclease I to predict mutation rates is also recommended37. Target sites with high genomic deletion rates can be selected through these three methods, and therefore, the efficiency of gene editing in B. dorsalis could be increased.
The development stage of the embryo determines whether mutations will be inherited. In order to obtain inherited mutations, gene editing must occur in germ cells. Generally, the mixture of sgRNA and Cas9 cannot be delivered into germ cells after pole cell formation has occurred. In B. dorsalis, the pole cell formation occurred at 3 h after egg laying38, whereas the protocol detailed here for B. dorsalis takes 30 min from embryo collection to the end of microinjection. During injection, nearly no pole cells are formed in the vegetal end of the embryo; therefore, gene mutations obtained in the G0 should be efficiently inherited. The time consumed by microinjection is also less than the method mentioned in previously published papers (generally 1-3 h)18,19.
It is important to minimize mechanical or chemical damage during the process of embryo collection and handling. Use a fine brush to gently line up embryos on the injection plate. The position of injection should reflect the work previously done in Drosophila (Methods – Nicolas Gompel's lab, http://gompel.org/methods) and Ceratitis capitata39. Inject embryos at the vegetal pole; never insert the needle into the animal pole. Prepare the needle following the micropipette puller guide; the opening of the needle should be as small as possible to avoid excessive cytoplasmic backflow. The method mentioned in published studies in B. dorsalis generally dechorionated the embryos with sodium hypochorite17,18,19; this could cause chemical damage and decrease the survival rates of the embryos. In this protocol, embryos are not dechorionated, and injection still works well. The chemical damage to the embryos could be minimal.
Post-injection insect rearing is very important. The standardized rearing protocol provided here can serve as a reference for rearing other fruit fly species, especially Bactrocera species. The embryos can be immersed in oil to avoid desiccation during injection using our self-made injection plate. Minimize the time larva spend in the oil (<2 h) to achieve survival rates in the G0 above 50%.
A non-invasive method of genomic DNA extraction is recommended for mutant detection. For example, some lepidopteran researchers suggest that the exuviates of the final instar larvae could be used to extract genomic DNA for further genotyping. For B. dorsalis, one non-invasive method involves extracting genomic DNA from a fresh puparium. Injection of multiple sgRNAs targeting a marker gene and the gene of interest could also improve the identification of mutants. For example, co-injected sgRNAs targeting eye color (kmo) and juvenile hormone receptor (Met) can produce 75% of offspring with double mutations in mosquitos. Met mutants were preliminarily screened based on the eye color of larvae37. This should be evaluated in B. dorsalis in the future to further improve the efficacy of gene editing in this insect species by using the CRISPR/Cas9 system.
In conclusion, the CRSIPR/Cas9 system is a powerful tool in functional genomics in B. dorsalis. Our detailed protocols provide useful information to help researchers to achieve efficient embryo collection, ideal survival rates of larvae, and the desired editing efficiency. This could be a simple and quick way to help researchers to obtain mutagenesis in B. dorsalis. This technique could not be applied in the functional studies of lethal genes since the inherited mutations need successful cross-breedings. Future studies could focus on developing transgenic tools to express stage or tissue-specific CRISPR elements to break through this limitation.
The authors have nothing to disclose.
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).
6x DNA Loading Buffer | TransGen Biotech | GH101-01 | |
Artificial climate chamber | ShangHai BluePard | MGC-350P | |
AxyPrep Genomic DNA Mini-Extraction Kit | Axygen | AP-MN-MS-GDNA-250G | |
BLAT | NA | NA | For searching potential gene loci in the genome |
Capillary Glass | WPI | 1B100F-4 | |
Eppendorf InjectMan 4 micromanipulator | Eppendorf | InjectMan 4 | |
GeneArt Precision gRNA Synthesis Kit | Thermo Fisher Scientific | A29377 | |
Hisat2 | NA | NA | For aligning the transcriptome to the acquired gene loci |
IGV | NA | NA | For visualizing the results from Transdecoder |
Microgrinder | NARISHIGE | EG-401 | |
Olympus Microscope | Olympus Corporation | SZ2-ILST | |
pEASY-Blunt Cloning Kit | TransGen Biotech | CB101-02 | https://www.transgenbiotech.com/data/upload/pdf/CB101_2022-07-14.pdf |
Phenol red solution | Sigma-Aldrich | P0290-100ML | |
Pipette cookbook 2018 P-97 & P-1000 Micropipette Pullers | Instrument Company | https://www.sutter.com/PDFs/cookbook.pdf | |
PrimeSTAR HS (Premix) | Takara Biomedical Technology | R040A | |
SAMtools | NA | NA | For generating the sorted bam files |
sgRNAcas9-AI | NA | NA | sgRNA design http://123.57.239.141:8080/home |
Sutter Micropipette Puller Sutter | Instrument Company | P-97 | |
Trans2K DNA Marker | TransGen Biotech | BM101-02 | |
Transdecoder | NA | NA | For combining the results of assemble transcripts and gene loci information https://github.com/TransDecoder/TransDecoder/releases/tag/TransDecoder-v5.5.0 |
TrueCut Cas9 Protein v2 | Thermo Fisher Scientific | A36498 | |
Ultra-trace biological detector | Thermo Fisher Scientific | Nanodrop 2000C |