Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9

The revolutionary RNA-guided endonuclease CRISPR/Cas9 system has proven to be a powerful tool for gene editing in a plethora of organisms. Here, utilizing this system we developed an efficient protocol for the generation of heritable germline mutations in the parasitoid jewel wasp, Nasonia vitripennis, a rising insect model organism for the study of evolution, development of axis pattern formation, venom production, haplo-diploid sex determination, and host–symbiont interactions. To establish CRISPR-directed gene editing in N. vitripennis, we targeted a conserved eye pigmentation gene cinnabar, generating several independent heritable germline mutations in this gene. Briefly, to generate these mutants, we developed a protocol to efficiently collect N. vitripennis eggs from a parasitized flesh fly pupa, Sarcophaga bullata, inject these eggs with Cas9/guide RNA mixtures, and transfer injected eggs back into the host to continue development. We also describe a flow for screening mutants and establishing stable mutant strains through genetic crosses. Overall, our results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in N. vitripennis, with strong potential for expansion to target critical genes, thus allowing for the investigation of several important biological phenomena in this organism.

2 with sgRNAs. Be sure to use nuclease-free consumables including filter tips and microfuge tubes. Also, thoroughly clean work area, microinjection apparatus, gloves, and pipettes with RNaseZap (Ambion) before conducting experiments.
• Once sgRNAs are produced, immediately mix these reagents with Cas9 protein at the final desired injection concentrations and make small 5-10ul aliquots. Store these readyto-inject final mixtures at -80°C until needed. The goal here is to avoid excess freezethaw-cycles for both the sgRNAs and the Cas9 protein as much as possible.
• Given that not all sgRNAs function efficiently, and specificity and activity are unpredictable, we recommend designing multiple sgRNAs for each target gene to increase probability of generating desired modifications of target genes.
• To collect enough N. vitripennis eggs for microinjection, it is important to expand stock wasp colonies to sufficient numbers. We recommend to establish 2-3 colonies with 200-500 wasps (of both sexes mixed) per colony.

Equipment
We recommend the following equipment, although each item can be substituted by others, depending on a given lab's set-up.

Genomic DNA target site design and selection criteria
The revolutionary CRISPR/Cas9 gene editing system relies on the target sequence encoded by an engineered sgRNA to guide the Cas9 nuclease to a desired genomic target location, allowing for base-pairing interactions between the sgRNA/Cas9 complex and the complementary genomic DNA sequence, thereby resulting in subsequent Cas9 mediated cleavage of the specified genomic target sequence. The characterized recognition sequence for the Streptococcus pyogenes Cas9 protein relies on the presence of a protospacer associated motif, or PAM, to be located 4 immediately adjacent to the desired genomic target sequence [4][5][6][7] . The PAM sequence is NGG, which is located in the genome directly downstream of the desired 20bp genomic target sequence, taking the form N20-NGG. Importantly, the PAM sequence is not included in the either the sgRNA template DNA, or the in vitro transcribed sgRNA. To define putative sgRNA genomic target sites we suggest to consider several factors. First, confirming transcriptional expression of the target region, and looking for conservation between other species, will help define putative N. vitripennis genomic target regions that have a higher probability of being necessary for gene function (assuming the goal is to disrupt gene function). To do this we recommend using both available N. vitripennis transcriptional databases and simple NCBI-BLAST searches (www.vector.caltech.edu) 8,9 . Secondly, once general target regions are defined, the putative sgRNA target sites can be identified by simply scanning both the sense and antisense strands for the presence of the NGG-PAMs either manually by eye, or by utilizing available software such as CHOPCHOP v2 10 , and/or local sgRNA Cas9 packages 11 . Finally, to minimize potential off-target effects, we recommend confirming specificity of the sgRNAs using publicly available bioinformatic tools for example NCBI-Blast 12 , Blat 13 and selecting the most specific sgRNAs within the specified target regions with the least potential off-target binding sites. It should be noted that even if the chosen sgRNA target sequences fulfill all of the above requirements, sgRNA specificity and activity is unpredictable. Therefore, we recommend that multiple different sgRNAs are designed to target the exonic coding sequences and co-injected in order to increase the chance of editing a target gene of interest.  Importantly, the first two nucleotides transcribed by the T7 RNA polymerase should ideally be "GG," and therefore, it is important that the in vitro transcribed sgRNA sequences beings with these two nucleotides. Therefore, if perhaps the chosen sgRNA sequences (green) do not begin with "GG," these nucleotides can be added onto the 5' end of the target sequence. For example, for the target sequences highlighted in figure 1, the following bases (pink) would be added to ensure robust in vitro transcription by the T7 RNA polymerase. For target # 1, two GG's would need to be added, for target # 2 no extra GG's would need to be added since it already begins with a GG, and for target # 3 one G would need to be added to ensure the first two nucleotides (underlined) begin with "GG." • Target # 1: GG ACATTACATCGGAATCGTACCGG The first step in sgRNA synthesis is to produce the template DNA to be used for in vitro transcription. This DNA is generated by template-free PCR using two primers that anneal to each other via complementary sequences (bold and underlined). These primers can be ordered from IDT as PAGE-purified oligos.
sgRNA-R (Table S1) -This is a universal reverse primer that can be used to generate all sgRNA targets containing the sgRNA backbone sequence.
sgRNA-F -This primer contains the T7 promoter upstream (orange) of the target sequence.

3'
Note: The GGN 20 is a generic sequence that includes the 20 nucleotide user-defined genomic target sequence (N 20 ), and the "GG" sequence necessary for in vitro T7 RNA polymerase transcription, but does NOT include the PAM sequence.
1. The first step in sgRNA production is to setup a template-free PCR using sgRNA-R and sgRNA-F primers to produce the linear dsDNA templates that will be used for in vitro transcription reactions. We prefer to use NEB's Q5 high-fidelity DNA polymerase, however other DNA polymerases should also work. Below is the PCR reaction we recommend. (11000 x g), discard the flow-through.

PCR Reagents
f. Repeat the step e again.
g. Add 20 ul nuclease-free water directly to the column matrix.
h. Incubate at room temperature for 1 min.
i. Transfer the column to a new 1.5 tube and centrifuge for 1 min (11000 x g) to elute the DNA.

4.
Following purification, measure the purity and concentration of the purified DNA template using a nanodrop. We aim to have a concentration of over 100 ng/ul to ensure enough template for the in vitro transcription reaction.

sgRNA production by in vitro transcription
To produce the sgRNAs, we use the Ambion MegaScript T7 in vitro transcription kit and followed the manufacturer's protocol.

1.
Briefly, we thaw and mix thoroughly the ribonucleotides (keep on ice) and reaction buffer (keep at room temperature), then add all reagents to a PCR tube in the following order.

In vitro Transcription Reagents Volume Reaction
Nuclease free water i. Place the filter cartridge into a new 1.5 ml tube.
j. Add 50 ul of nuclease-free water to the center of the filter cartridge.
k. Close the cap of the tube and incubate at 70°C for 10 min.
l. Centrifuge (13000 x g) for 1 min at room temperature to elute RNA.
6. The final concentration should be measured using a nanodrop, and quality can be measured with an Agilent Bioanalyzer confirming that sgRNA appears as a single band without any degradation products.
7. sgRNAs can then be diluted to 1000 ng/ul in nuclease-free water and stored in aliquots @ -80°C. We generally produce roughly 5-100ug of sgRNA from this reaction depending on the template DNA quality.

Preparation of sgRNA/Cas9 mixtures for microinjection
Before microinjection the purified recombinant Cas9 protein from Streptococcus pyogenes should be obtained commercially (CP01, PNA Bio Inc) and diluted to 1000 ng/ul using UltraPure DNase/RNase-free distilled nuclease free water and stored @ -80°C.
• This stock Cas9 protein solution should be diluted with nuclease free water and mixed with the purified sgRNAs at various concentrations (20-320 ng/ul) in small 5-10ul aliquots.
• These ready-to-inject final mixtures can be stored at -80C until needed. The goal here is to avoid excess freeze-thaw-cycles for both the sgRNAs and the Cas9 protein as much as possible.
• For N. vitripennis, we found the optimal concentrations for both the Cas9 protein and purified sgRNAs to be 160 ng/ul for each component.
• To prepare these mixtures thaw and mix both components in UltraPure DNase/RNasefree distilled nuclease free water on ice, and maintain these mixtures on ice while performing injections.

Preparation of needles for N. vitripennis embryo microinjection
For effective penetration and microinjection into N. vitripennis eggs, we experimented with

5.
Allow female wasps to parasitize (oviposit embryos) the host for roughly 30 minutes at 25℃. Then remove the host and replace with a new host, every 15 minutes, to ensure sufficient eggs for continuous injection. Note -it is very important that the embryos are as young as possible, ideally within the first hour of being oviposited, to ensure that they are in the pre-blastoderm stage. Old embryos (>1.0 hour) should not be injected.

6.
To collect embryos, remove parasitized hosts from the foam stopper. Under a dissecting microscope, carefully peel off the posterior end of the puparium that was exposed to the wasps using forceps. Embryos will be resting on the surface of the host pupa ( figure 4).
Carefully remove embryos from host, using a fine-tip wet paintbrush, ensuring not to burst the soft pupal skin inside the host. Note: Ultrafine tweezers can also be used to gently pick off the embryos; however, care must be taken in this particular case to not 14 burst the embryos or the host pupa.

7.
Transfer embryos one-by-one to double-sided sticky tape (fixed to a glass slide). Using a wet paintbrush (or single side of a pair of ultrafine forceps) orient the eggs one-by-one in a row so the posterior end (more narrow end) is pointing in the same direction for each egg ( figure 5). Note -we found embryo survival rates to be greater if we did not cover eggs with halocarbon oil during injection as is done for Drosophila melanogaster microinjection 15 . Since oil is not used, it is important to keep the embryos moist during the injection period by regularly adding water using the paintbrush. The amount of water 15 on the brush is key to move embryos around and align with ease. Too much water results in embryos floating and not sticking well to the double-sided sticky tape, and too little water makes them difficult to move around and can lead to desiccation. To adjust the degree of moisture, dip the tip of the brush in water then lightly touch the tip of the brush to a dry kimwipe.

4.
Inject ~40 eggs at a time (should take roughly 10 minutes) then stop and transfer injected eggs into a host then continue injecting again using a fresh newly laid batch of eggs.
Note: Depending on needle, needle clogging will likely be an issue. In our experience, the needle would clog once in every 25 embryo injections, and we would either re-bevel the needle or would need to use a new needle.
Transferring embryos back to the hosts 1. Following microinjection, transfer injected embryos back into a pre-stung Sarcophaga bullata pupae using a fine-tip paintbrush (figure 6) or ultrafine forceps. N. vitripennis larva utilize the host pupa as a food source to complete larval development and to our knowledge there is currently no available artificial diet that can be used.

2.
Very important -be sure to only transfer eggs back into a pre-stung host, otherwise embryos will not survive. When a female wasp stings a host, she uses her ovipositor to bore a hole in the host puparium to inject venom which causes arrest of the pupal development and begin tissue necrosis, allowing the N. vitripennis larvae to consume the host. Without the venom, the host will survive and the N. vitripennis larvae will not be able to consume the host.

3.
To ensure a host has already been stung, find a host with embryos in it, then scrape all the embryos off and use it as the host. Also, to avoid overcrowding, only place about 40 injected embryos or less per host.

4.
Incubate hosts harboring transferred injected eggs in a moist humidified chamber (e.g. petri dish with cotton balls moist with water) at 25°C until hatching (roughly 1-2 days).
Importantly, hosts can be left with a peeled off puparium and the N. vitripennis eggs will develop normally so long as they are incubated in a humidified chamber (petri dish with damp filter paper and cotton balls) with roughly 70% relative humidity (figure 7) .

5.
Monitor the embryos, the hatched N vitripennis larvae, and the host daily. Remove any dead N. vitripennis larvae, and if the host becomes infected with bacteria or dies (turns to the gray or dark color and has a foul smell) transfer the larvae to a fresh pre-stung host.
Genetic crosses and screening for mutations 1. After roughly 8 days the injected embryos will begin to pupate. Once they pupate they will no longer consume food (i.e. blowfly host) and can be removed from the host.

2.
Remove each N. vitripennis pupae from the host, and place in an individual 1.5ml eppendorf tube until hatching. This will ensure that the hatched females will be virgin and will not mate until desired.  o. Centrifuge for 1 min at 8000 x g.

5.
The presence of mutations can be determined by PCR amplifying/sequencing the 20 genomic target region.

6.
Colonies that have mutations as determined by sequencing should be continued, while colonies that were established with non-mutant G0's should be discarded.

7.
Importantly, unmated females will give rise to 100% haploid male broods, so therefore a mutant unmated female can give rise to large number of mutant males that can be used for subsequent analysis. Note: mutations in essential genes will need to be kept by mating surviving G0 injected females (presumably heterozygous for a mutation) to wild type males; in this particular case, half of the F1 male progeny should die due to inheritance of the lethal mutation, while half of the F1 female progeny will be carriers of the lethal mutation. Table S1. Primers used in this study.