1. N. Vitripennis Colony Rearing
2. Collection and Alignment of N. Vitripennis Pre-blastoderm Stage Embryos
3. Needle Preparation for Microinjections
4. Embryo Microinjection
5. Transplanting Injected G0 N. vitripennis Embryos onto Pre-stung Host
6. Screening for Genome Modifications
This protocol provides detailed guidelines for colony rearing, pre-blastoderm embryo collection, alignment, microinjection, and subsequent transplantation after injection and can be used for efficient genome engineering in N. vitripennis. As shown in Figure 2, the general sequence of steps for a successful microinjection into N. vitripennis include: (i) permitting male and female adults to mate (~ 4 days), (ii) supplying fresh host fly pupae (S. bullata) placed in a modified foam plug to mated females and allowing for oviposition (~ 45 min), (iii) carefully peeling away the parasitized host pupae cuticle to expose and collect pre-blastoderm stage wasp embryos (~ 15 min), (iv) aligning collected embryos (~ 15 min), (v) microinjecting genome modification components into embryos (~ 15 min), (vi) carefully placing injected embryos back into pre-stung hosts to allow for proper development (~ 15 min), and (vii) preventing dehydration of the injected embryo/host by transferring them into a humidified chamber with roughly 70% relative humidity (~ 15 min). Parasitized hosts are then incubated for roughly 14 days to allow for complete development of N. vitripennis embryos. Once injected, adults emerge from the host (viii), isolate, mate, and screen them individually for the presence of expected mutations.
For effective needle penetration and microinjection into N. vitripennis embryos, several types of capillary glass needles with filament including quartz, aluminosilicate, and borosilicate types are tested. It is found that the quality of needle is critical for avoiding breakage/clogging during injection, and for achieving high rates of both embryo survival and transformation efficiency. For each glass type, an effective protocol was developed to pull needles in order to have a desired hypodermic-like long tip effective for N. vitripennis embryo microinjection using different micropipette pullers (P-1000, and P-2000) (Table 1, Figure 4).
To optimize the procedure, survival rates are measured following injection of varying amounts of genome modification components. The genome modification components used here were mixed guide RNAs and Cas9 protein for CRISPR-mediated genome editing, which were demonstrated previously to work well in N. vitripennis21. Similar to what was previously reported, here a sgRNA targeting the cinnabar gene is designed and synthesized. By targeting and disrupting this gene, an easily identifiable phenotypic change is seen in the eye color of the organism19,21. An injection mixture combining a variety of concentrations of sgRNA (0, 20, 40, 80, 160, and 320 ng/µL) with Cas9 protein (0, 20, 40, 80, 160, and 320 ng/µL) is created and injected into embryos of wild type N. vitripennis. Survival rate of injected embryos is found to be dose-dependent (Table 2)21. The increased concentration of sgRNA and Cas9 protein lead to decreased survival rates (Table 2), perhaps due to additive off-target effects. High humidity (~ 70%) is also found to be important for embryo survival after transplantation to hosts, as low humidity (~ 10%) resulted in 100% death to all injected embryos.
Figure 1. Preparation of host pupae (S. bullata) for N. vitripennis embryo oviposition. (A)Young and old S. bullata pupae. Older pupae have a darker cuticle whereas younger pupae have a more reddish tint to their cuticle. Younger pupae are preferred for maximizing oviposition. Posterior and anterior ends of the pupae can also be distinguished by a "crater-like opening on the posterior end, whereas the anterior end comes to a rounded point. (B) Host (S. bullata) pupa preparation for N. vitripennis embryo oviposition. Inserting the host pupae into a foam plug that has had a pupae sized hole carved out. Have the posterior side of the host pupae face inside the plug while 0.2 cm of the anterior end exposed to allow for maximum concentration of oviposition into the anterior area. Please click here to view a larger version of this figure.
Figure 2. Timeline for creating N. vitripennis mutants by microinjection. Timeline of N. vitripennis embryo collection, CRISPR/Cas9 microinjection, and post-injection procedures. N. vitripennis adults were allowed to mate in absence of an oviposition site for 4 days (i). Following, a fresh, flesh fly host pupae, S. bullata, placed inside a foam stopper as to only expose 0.5 cm of the posterior end, was introduced to the gravid females for 45 min to allow for parasitization (ii). Concurrently injection materials including microinjection needles and CRISPR/Cas9 components were prepared (iii). Embryos were collected from the host (iv), aligned (v), and injected with CRISPR/Cas9 components (vi). The injected embryos were carefully transferred back to a pre-stung host (vii) and incubated till fully developed (14 days) (viii). When the adults emerged, mutants were screened for phenotypes of expected CRISPR/Cas9 induced mutations in the target gene (ix). The entire procedure generally takes 19 days to complete. Please click here to view a larger version of this figure.
Figure 3. Modified microscope slide for lining embryos. (A) A coverslip. (B) A microscope slide. (C) An embryo alignment device. A coverslip can be glued onto a microscope slide to be used to line embryos for injection. The purpose of the coverslip is to act as an edge to allow for easy manipulation and lining of embryos. Please click here to view a larger version of this figure.
Figure 4. Injection needle preparation. (A) Examples of good and bad aluminosilicate glass needles. (B) The tips of an unbeveled, correctly beveled, and poorly beveled needle. Aluminosilicate glass capillary tubes were pulled by using a micropipette puller. The produced needle tips were then gently opened and refined using a beveler. The good bevelled needle has a very sharp tip, and the bad bevelled needle has a blunt tip. Please click here to view a larger version of this figure.
Capillary Glass Type | Sutter Needle Puller Model | Heat | Filament | Velocity | Delay | Pull | Pressure |
Quartz | P-2000 | 750 | 4 | 40 | 150 | 165 | – |
Aluminosilicate | P-1000 | 605 | – | 130 | 80 | 70 | 500 |
Borosilicate | P-1000 | 450 | – | 130 | 80 | 70 | 500 |
Table 1. Settings for needle puller.
Note: Needle puller setting vary from machine to machine so each lab will need to optimize their own needle puller settings. This table has been modified from Li et al.21
sgRNA-1 | Cas9 | Total embryos | Transplantation (10% humidity) | Transplantation (70% humidity) | |||
Larvae Survivors | Larvae Survivors | Adult survivors | |||||
Total (%) | Total (%) | ♂ | ♀ | Total (%) | |||
No injection | No injection | 100 | 94 (94) | 96 (96) | 66 | 26 | 92 (92) |
Water | Water | 100 | 0 (0) | 78 (78) | 44 | 32 | 76 (76) |
20 ng/µL | 20 ng/µL | 100 | 0 (0) | 74 (74) | 34 | 34 | 68 (68) |
40 ng/µL | 40 ng/µL | 100 | 0 (0) | 67 (67) | 30 | 32 | 62 (62) |
80 ng/µL | 80 ng/µL | 100 | 0 (0) | 53 (53) | 24 | 22 | 46 (46) |
160 ng/µL | 160 ng/µL | 100 | 0 (0) | 41 (41) | 16 | 22 | 38 (38) |
320 ng/µL | 320 ng/µL | 100 | 0 (0) | 25 (25) | 10 | 10 | 20 (20) |
Table 2. Injection and transplantation survivorship and mutagenesis rates based on injections of different concentrations of sgRNA and Cas9. This table has been modified from Li et al.21
Bugdorm | Bugdorm | 41515 | Insect Rearing Cage |
Glass Test Tubes | Fisher Scientific | 982010 | |
Flesh fly pupae, Sarcophaga bullata | Carolina Insects | 144440 | |
Microelectrode Beveler | Sutter Instruments | BV10 | |
Diamond abrasive plate (0.7u to 2.0u tip sizes) | Sutter Instruments | 104E | |
Micromanipulator | World Precision Instruments | Kite R | |
Femtojet Express programmable microinjector | Eppendorf | ||
Micropipette Puller | Sutter Instruments | P-1000 or P-2000 | |
Stereo Microscope | Olympus | SZ51 | |
Compound Microscope | Leica DM-750 | ||
Aluminosilicate glass capillary tubing 1mm(outside diameter) X 0.58mm (inner diameter) | Sutter Instruments | BF100-58-10 | Can also use Borosilicate or Quartz |
Identi-Plugs | JAECE | L800-B | Foam plugs |
Microscope Slides | Fisherbrand | 12-550-A3 | |
Micro Cover glass | VWR | 48366045 | |
Adhesive | Aron Alpha | AA471 | For glueing coverslip onto microscope slide |
Fine-tip paintbrush | ZEM | 2595 | |
Ultra-fine tip forcep | Fisher Scientific | 16-100-121 | |
Femtotips Microloader tips | Fisher Scientific | E5242956003 |
The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.
The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.
The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.