Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

Summary

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

Abstract

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

Introduction

Gene editing technologies could be used to introduce insertions or deletions into a specific genome locus to artificially modify the target gene on purpose¹. In the past years, CRISPR/Cas9 technology has developed rapidly and has a growing scope of applications in various fields of life sciences^2,3,4,5,6. The CRISPR/Cas9 system was discovered back in 1987⁷, and widely found in bacteria and archaea. Further research indicated that it was a prokaryotic adaptive immune system that depends on the RNA-guided nuclease Cas9 to fight against phages⁸. The artificially modified CRISPR/Cas9 system mainly consists of two components, a single guide RNA (sgRNA) and the Cas9 protein. The sgRNA is made up of a CRISPR RNA (crRNA) complementary to the target sequence and an auxiliary trans-activating crRNA (tracrRNA), which is relatively conserved. When the CRISPR/Cas9 system is activated, the sgRNA forms a ribonucleoprotein (RNP) with the Cas9 protein and guides Cas9 to its target site via the base pairing of RNA-DNA interactions. Then, the double-strand DNA can be cleaved by the Cas9 protein and as a result, the double-strand break (DSB) emerges near the protospacer adjacent motif (PAM) of the target site^9,10,11,12. To mitigate the damage caused by the DSBs, cells would activate comprehensive DNA damage responses to efficiently detect the genomic damages and initiate the repair procedure. There are two distinct repair mechanisms in the cell: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is the most common repair pathway that can repair DNA double-strand breaks quickly and prevents cell apoptosis. However, it is error-prone because of leaving small fragments of insertions and deletions (indels) near the DSBs, which usually results in an open reading frame shift and thus can lead to gene knock-out. In contrast, homologous repairment is quite a rare event. On the condition that there is a repair template with sequences homologous to the context of the DSB, cells would occasionally repair the genomic break according to the nearby template. The result of HDR is that the DSB is precisely repaired. Especially, if there is an additional sequence between the homologous sequences in the template, they would be integrated into the genome through HDR, and in this way, the specific gene insertions could be realized¹³.

With the optimization and development of the sgRNA structures and Cas9 protein variants, the CRISPR/Cas9-based genetic editing system has also been successfully applied in research of insects, including but not limited to Drosophila melanogaster, Aedes aegypti, Bombyx mori, Helicoverpa Armigera, Plutella xylostella, and Locusta migratoria^{14,15,16,17,18,19}. To the best of the authors' knowledge, although RNPs consisting of the Cas9 protein and in vitro transcribed sgRNA have been used for locust genome editing^20,21,22, a systematic and detailed protocol for CRISPR/Cas9 ribonuclease mediated construction of homozygous mutants of the migratory locust is still lacking.

The migratory locust is an important agricultural pest that has a global distribution and poses substantial threats to food production, being especially harmful to gramineous plants, such as wheat, maize, rice, and millet²³. Gene function analysis based on genome editing technologies can provide novel targets and new strategies for the control of migratory locusts. This study proposes a detailed method for knocking out migratory locust genes via the CRISPR/Cas9 system, including the selection of target sites and design of sgRNAs, in vitro synthesis and verification of the sgRNAs, microinjection and culture of eggs, estimation of mutation rate at the embryonic stage, detection of mutants as well as passage and preservation of the mutants. This protocol could be used as a basal reference for manipulation of the vast majority of locust genes and can provide valuable references for genome editing of other insects.

Protocol

1. Target site selection and sgRNA design

1. Collect as much sequence information as possible for the gene of interest through literature research and/or searching for the mRNA or coding DNA sequence (CDS) of the gene at NCBI and locust database²⁴.
2. Compare the sequence of the interested gene to its genomic DNA sequence to distinguish the exon and intron regions.
3. Select a candidate region for target site design based on the research purpose. Design primer pairs to amplify the candidate region fragment and verify its wild-type sequence by sequencing analysis of the PCR product.
   NOTE: There are different strategies for the selection of the candidate target region. For example, in most gene/protein function research, use the exon fragment close to its start codon as the candidate region to ensure that the whole protein/RNA function is lost after the genome editing (i.e., gene knock-out). While for the function analysis of a specific domain, select the candidate region at the beginning (or both ends) of the domain.
4. Use online resources such as the E-CRISP Design²⁵ to search for potential target sites in the candidate target region.
   1. Select "Drosophila melanogaster BDGP6" (or any other insect) in the drop-down list of reference genomes and choose "Input is FASTA sequence" followed by pasting the sequence of target region into the textbox (in FASTA format).
   2. Start the application by pressing "Start sgRNA search" with "medium" and "single design" to get the possible target sites. Select 1-3 potential targets from the results based on their predicted scores and design the corresponding sgRNAs accordingly.
      NOTE: There are many more online platforms for CRISPR design, such as CRISPOR, CHOPCHOP, CRISPRdirect, ZiFiT, and so on (see Table of Materials). Some of them have the function of specificity check for the species whose genomic sequence data is in their databases. However, the genomic sequence of locusts has not been found on any online website. It is better to use more than one online tool for the CRISPR design in locusts. Comprehensively, consider the results from different websites and select the sgRNA from the results on the principle of highest specificity, highest efficiency, and least mismatch rate (Figure 1A,B).

2. Synthesis and verification of the sgRNA  in vitro

1. Synthesize the sgRNA using sgRNA synthesis kits according to the manufacturer´s manual (Table of Materials). This procedure usually includes three steps: DNA template amplification, in vitro transcription, and sgRNA purification²¹. Dilute the synthesized sgRNA with nuclease-free water to a storage concentration of 300 ng/µL for further use.
2. Amplify and purify the target gene fragment to serve as the substrate of the in vitro cleavage assay of the CRISPR/Cas9 system. Perform these steps following the manufacturer's instructions.
3. Dilute the purchased Cas9 protein to 300 ng/µL with nuclease-free water. Incubate 1 µL of sgRNA (300 ng/µL) and 1 µL of Cas9 protein (300 ng/µL) with 200 ng of purified target fragment in the cleavage buffer at 37 °C for 1 h (10 µL of total volume; see Table 1). Estimate the efficiency of sgRNA induced Cas9 cleavage by agarose gel electrophoresis (Figure 1C). Select the sgRNAs with high activity for the following microinjection.
   NOTE: The results of agarose gel electrophoresis can be converted to grayscale images with image processing software and the cleavage efficiency is estimated according to the grayscale of the bands of speculated sizes.

3. Microinjection and culture of the eggs

1. Prepare the injection needles by pulling glass capillaries with a micropipette puller (Table of Materials). Set the parameters as follows: Heat to 588, Pull to 90, Velocity to 60, and Time to 40. Grind the tip of the injection needle with a micro grinder (Table of Materials).
   NOTE: Ideal needle tips are open and sharp-edged (Figure 2A). It is highly recommended to prepare additional needles for the experiments because needles are sometimes blocked or accidentally broken during injections.
2. Mix 1 µL of Cas9 protein (300 ng/µL) with 1 µL of the verified sgRNA (300 ng/µL) together to obtain the RNPs for injection. Add 8 µL of RNase-free sterile water to make the final volume of the mixture to 10 µL. Mix the solution thoroughly and keep it on ice.
   NOTE: The final concentrations of the Cas9 protein and sgRNAs can be optimized according to the editing results. Avoid repetitive freezing and thawing of the prepared RNP solution and immediate use is recommended.
3. Rear the male and female adult locusts together at 30 °C with a 16 (light):8 (dark) photoperiod and supply them sufficient fresh wheat seedlings as food. Observe these locusts daily and put an oviposition pot (a plastic flowerpot or cup filled with wet sterile sand) into the rearing cage for oviposition once the locusts mated.
   NOTE: Usually, 100 pairs of adult locusts reared in a rearing cage (40 cm x 40 cm x 40 cm) are enough to generate eggs for knocking out a single gene. Culture additional locust pairs if more eggs are needed.
4. Collect the freshly-laid egg pods from the oviposition pot and wait for about 30 min for egg tanning. Gently isolate the eggs from the egg pods in water using a fine brush and wash them with sterile water three times. Keep the eggs in a Petri dish and add sterile water to keep them moist.
   NOTE: Tanning of the newly-laid eggs can significantly improve the mutant efficiency²¹.
5. Fill an injection needle with the prepared RNP solution and load it to the micromanipulator (Table of Materials).
   1. Set the microinjection parameters as follows: 300 hPa of the injection pressure (pi), 0.5 s of the injection time (ti), and 25 hPa of the compensation pressure (pc).
   2. Press the pedal to evaluate the volume of the injected solution. Adjust the injection pressure and injection time to ensure that the injection volume is about 50-100 nL.
      NOTE: Exhaust the residual air in the needle to make the injection solution continuous and controllable as much as possible. The connection of the needle and the micromanipulator need to be tight.
6. Arrange the sterile eggs regularly on the injection pad (Figure 2B,C) and place the pad on the object table of the microscope (Figure 3A). Adjust the magnification of the microscope until the eggs are observed. Adjust the microinjection needle under the microscope until the injection tip can be seen and position it near the egg to be injected.
7. Start the injection at a suitable height and an angle of 30-45 degrees. Insert the tip into the egg carefully near the micropyles of the egg (Figure 3B) and press the pedal to accomplish the injection. Retract the needle quickly and move the injection pad for injection of the next egg.
   NOTE: A slight expansion of the egg during the microinjection should be observed. A small amount of cytoplasmic leakage at the pinhole is acceptable. Replace the injection needle with a new one or adjust the injection angle if the outflow of fluid is too much.
8. Transfer the injected eggs to a culture dish (a Petri dish with a piece of moist filter paper), and place them in an incubator at 30 °C.
   NOTE: It will take about 13-14 days till nymphs hatch from these injected eggs (on condition that mutation of the target gene does not affect the development of the embryos) (Figure 3C). Inject at least 100 eggs to ensure a sufficient hatching and eclosion amount for knocking out a specific gene.

4. Mutation rate estimation and the screening of mutants

1. Check the development of the injected eggs every day after the injection. Transfer injected eggs to a new culture dish every 24 h in the first 5 days.
2. On the 6^th day (or later) after injection, randomly pick and transfer 10 eggs into a tube and adequately grind the eggs with two steel balls at 60 Hz for 6 min using a grinder (see Table of Materials). Resuspend the debris with 1 mL of PBS. Transfer 5 µL of the mixture into 45 µL of 50 mM NaOH and lyse at 95 °C for 5 min. Add 5 µL of 1 M Tris-HCl (pH=8.0) into the lysis system to terminate the alkaline lysis reaction.
   NOTE: Eggs can be picked out for alkaline lysis on any day after the last transfer and multiple eggs can be used as one sample depending on their development situation (e.g., five 6-day-old embryos can be used as one sample, and two 10-day-old embryos can be used as one sample). Sometimes, it is not feasible to get the genomic DNA of eggs using the alkaline lysis method. Commercial genomic DNA extraction kits can be used as an alternative method for isolating the genomic DNA from the eggs.
3. Take 1 µL of the lysis product as the PCR template to amplify the target gene fragment (Table 2 and Table 3) and send the PCR products for sequencing. Compare the sequencing results with the wild-type sequence to preliminarily evaluate whether the CRISPR/Cas9 system cleaved the target gene in vivo (Figure 4A). Allow the rest of the eggs for subsequent development on the condition that indels are detected at the embryonic stage.
   NOTE: In this way, the mutation rate can be preliminarily estimated at the embryo stage. If no indels are found at this step, prepare some new sgRNAs for the target gene and repeat the knock-out protocol from step 2.1.
4. Transfer the hatched nymphs to a rearing cage and culture them as described in step 3.3. When the nymphs developed to the fifth instar stage, separate them with plastic culture cups (1 nymph/cup).
   NOTE: It usually takes about 25-35 days for the nymphs to develop to their fifth instar and the most obvious phenotype is that the wing buds extend to the fourth or fifth abdominal segments. Moreover, it is recommended to record the phenotypes of dead nymphs to predict the relationship between the target gene and phenotypes in further research.
5. Cut off about 2 mm length of the antennae with dissecting scissors and lyse it using the alkaline lysis method (step 4.2). Analyze the target gene fragment sequence of these nymphs as described above (step 4.3) to identify the G₀ mutants (Figure 4B).
   ​NOTE: The antennae cut for lysis can be a little longer and grinding or mincing the antennae is helpful for lysis although the antennae can be directly digested. Individuals with multiple peaks near the target site in the sequencing result are identified as positive mutants and allowed for subsequent development and mating.

5. Establishment and passaging of mutant lines

1. Perform cross breeding using the G₀ mutants and wild-type locusts (Figure 4B). Collect the oocysts and incubate these oocysts separately at 30 °C. Use 3-6 developed eggs in each oocyst to detect mutations as described in steps 4.2 and 4.3. Keep mutation-positive oocysts for the subsequent development and abandon the mutation-negative oocysts.
   NOTE: Mutation rate estimation at the embryonic stage can greatly accelerate the screening of G₁ mutants. Usually, 3-6 developed eggs can be mixed as one sample for PCR-mediated genotyping as described above.
2. Rear the G₁ nymphs as described in step 4.4. Cut off about 2 mm length of the antennae to perform PCR-based genotyping as described in step 4.5 for detecting G₁ heterozygotes. Perform TA-cloning according to the manufacturer's instructions (see Table of Materials) to identify their mutations. Perform in-cross using G₁ heterozygotes with the same mutations to obtain G₂ nymphs.
   NOTE: Sanger sequencing of the PCR products can only provide information for finding out the heterozygotes, but not enough information for identifying the exact mutations. Thus, TA cloning using the PCR products is required to clearly identify the mutations and can promote the establishment of stable mutant lines.
3. Rear the G₂ nymphs till their fifth instar. Use the PCR-based genotyping described in step 5.2 to identify the homozygotes and/or heterozygotes that are suitable for further research and stable passaging (Figure 4B).
   ​NOTE: Pay attention to avoid mixing of homozygotes and heterozygotes. It is recommended to perform PCR-based genotyping in every generation to confirm the homozygosity and/or heterozygosity of each population. The number of locusts used for this check depends on the population size. Moreover, the population of locusts can be expanded by the in-cross strategy.

6. Egg cryopreservation and resuscitation

1. Wash the eggs to be cryopreserved with sterile water and incubate them in a culture dish as described above (step 3.8) for 5-6 days. Gather these eggs together in the culture dish and cover them with filter paper fragments. Wrap the entire culture dish with a paraffin film.
2. Keep the dish at 25 °C for 2 days followed by another 2 days at a relatively lower temperature (13-16 °C). Then, transfer the dish to a 6 °C refrigerator. Add water into it every 2 weeks to provide a moist environment for the eggs (Figure 5A).
   NOTE: The embryos are speculated to be at the katatrepsis stage after being developed for 5-6 days at 30 °C²⁶. Gradient cooling is helpful for cryopreservation of eggs (Table 5).
3. For resuscitating the cryopreserved eggs, take the Petri dish out from the refrigerator and keep it at 25 °C for 2 days. Place these eggs in a 30 °C incubator until the nymphs hatch (Figure 5B).
   NOTE: It is necessary to put the cryopreserved eggs at 25 °C before transferring them to a 30 °C incubator. The embryos can be cryopreserved for at least five months although the hatching rate and eclosion rate can decrease because of the cryopreservation (Table 5).

Results

This protocol contains the detailed steps for generating homozygous mutants of the migratory locusts with the RNP consisting of Cas9 protein and in vitro synthesized sgRNA. The following are some representative results of CRISPR/Cas9-mediated target gene knockout in locusts, including target selection, sgRNA synthesis and verification (Figure 1A), egg collection and injection, mutant screening and passaging, cryopreservation, and resuscitation of the homozygous eggs.

Discussion

Locusts have been among the most devastating pests to agriculture since the civilization of human beings²³. CRISPR/Cas9-based genome editing technology is a powerful tool for providing better knowledge of the biological mechanisms in locusts as well as a promising pest control strategy. Thus, it is of great benefit to develop an efficient and easy-to-use method of CRISPR/Cas9-mediated construction of homozygous locust mutants. Although some great works have been reported and provided some basic wo...

Materials

Name	Company	Catalog Number	Comments
10×NEBuffer r3.1	New England Biolabs	B7030S	The buffer of  in vitro Cas9 cleavage assays
2xEs Taq MasterMix (Dye)	Cwbio	CW0690	For gene amplification
2xPfu MasterMix (Dye)	Cwbio	CW0686	For gene amplification
CHOPCHOP			Online website for designing sgRNAs, http://chopchop.cbu.uib.no/.
CRISPOR			Online website for designing sgRNAs, http://crispor.org.
CRISPRdirect			Online website for designing sgRNAs, http://crispr.dbcls.jp/.
Electrophoresis power supply	LIUYI BIOLOGY	DYY-6D	Separation of nucleic acid molecules of different sizes
Eppendorf Tube	Eppendorf	30125177	For sample collection, etc.
Fine brushes	Annigoni	1235	For cleaning and isolating eggs. Purchased online.
Flaming/brown micropipette puller	Sutter Instrument	P97	For making the microinjection needles
Gel Extraction Kit	Cwbio	CW2302	DNA recovery and purification
Gel Imaging Analysis System	OLYMPUS	Gel Doc XR	Observe the electrophoresis results
GeneTouch Plus	Bioer	B-48DA	For gene amplification
Glass electrode capillary	Gairdner	GD-102	For making injection needles with a micropipette Puller
Incubator	MEMMERT	INplus55	For migratory locust embryo culture
Metal bath	TIANGEN	AJ-800	For heating the sample
Micro autoinjector	Eppendorf	5253000068	Microinjection of embryos early in development
Micro centrifuge	Allsheng	MTV-1	Used for mixing reagents
Microgrinder	NARISHIGE	EG-401	To ground the tip of injection needle
Microloader	Eppendorf	5242956003	For loading solutions into the injection needles.
Micromanipulation system	Eppendorf	TransferMan 4r	An altinative manipulation system for microinjection
Microscope	cnoptec	SZ780	For microinjection
Motor-drive Manipulator	NARISHIGE	MM-94	For controling the position of the micropipette during the microinjection precedure
Multi-Sample Tissue Grinder	jingxin	Tissuelyser-64	Grind and homogenize the eggs
ovipisition pot	ChangShengYuanYi	CS-11	Filled with wet sterile sand for locust ovipositing in it. The oocysts are collected from this container. Purchased online.
Parafilm	ParafilmM	PM996	For wrapping the petri dishes.
pEASY-T3 Cloning Kit	TransGen Biotech	CT301-01	For TA cloning
Petri dish	NEST	752001	For culture and preservation of  the eggs.
Pipettor	Eppendorf	Research®plus	For sample loading
plastic culture cup			For rearing locusts seperately and any plastic cup big enough (not less than 1000 mL in volume) will do. Purchased online.
Precision gRNA Synthesis Kit	Thermo	A29377	For sgRNA synthesis
Primer Premier	PREMIER Biosoft	Primer Premier 5.00	For primer design
SnapGene	Insightful Science	SnapGene®4.2.4	For analyzing  sequences
Steel balls	HuaXinGangQiu	HXGQ60	For sample grinding.Purchased online.
Tips	bioleaf	D781349	For sample loading
Trans DNA Marker II	TransGen Biotech	BM411-01	Used to determine gene size
TrueCut Cas9 Protein v2	Thermo	A36496	Cas9 protein
UniversalGen DNA Kit	Cwbio	CWY004	For genomic DNA extraction
VANNAS Scissors	Electron Microscopy Sciences	72932-01	For cutting off the antennae
Wheat			To generate wheat seedlings as the food for locusts. Bought from local farmers.
ZiFiT			Online website for designing sgRNAs, http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx.