Embryo Microinjection: A Technique to Deliver a Compound into the Zebrafish Yolk

Protokół

1. Microinjection of CRISPR-components into Zebrafish Embryos

1. Set up breeding tanks the night prior to injecting by placing the number of desired males and females (typically 2 females and 1 or 2 males) in a breeding tank with a divider in place.
2. Prepare a microinjection plate with 1.5% agarose in 1x E3 media (see Table of Materials) with 0.01% methylene blue (a fungicide) by pouring 35 mL of the melted agarose into a 10 cm Petri dish and gently lay a plastic mold to create wedge-shaped troughs into the solution, tapping the mold to eliminate air bubbles.
3. Allow the agarose to set, and store the dish with a small amount of media and wrapped in paraffin film to prevent the plate from drying out at 4 °C.
   NOTE: Injection plates are reusable for several weeks, until the wells become deformed or dry, or the plate begins to grow mold.
4. On the morning of injecting, thaw purified sgRNA and Cas9 protein on ice. Remember to handle all materials with gloves to prevent RNase contamination and to use RNase-free tips and tubes.
5. Generate a 5 µL injection solution by combining Cas9 protein and the sgRNA in a 2:1 ratio of Cas9:sgRNA to obtain final concentrations of 400 pg/nL Cas9 protein and 200 pg/nL sgRNA. Incubate the Cas9/sgRNA solution at room temperature for 5 min to allow the Cas9 and sgRNA to form a ribonucleoprotein complex. Add 0.5 µL of 2.5% wt/vol phenol red solution (see Table of Materials), and RNase-free water to a final volume of 5 µL.
   NOTE: The ionic strength of the solution has been shown to affect the solubility of the Cas9/sgRNA complex, therefore the addition of KCl may increase the cutting efficiency of sgRNAs that exhibit low indel formation.
6. Make an injection needle by pulling a 1.0 mm glass capillary using a micropipette puller. Cut the tip of the freshly-made needle using a new razor blade or forceps to obtain an angled opening that will easily pierce the chorion and yolk sac.
7. Place the needle in a micromanipulator attached to a microinjector with the air source turned on. Under a light microscope using the magnification suitable for the calibration determined for the particular apparatus, adjust the injection pressure until the needle consistently ejects a 1 nL solution into a Petri dish filled with mineral oil.
   NOTE: The quality of the needle is critical. Practice producing a needle and injecting into the yolk sac of embryos until this is skill is mastered before attempting further experiments.
8. Remove the divider and allow the fish to breed for approximately 15 min.
   NOTE: Longer breeding times will produce more embryos, however the injection should be completed while the embryos are at the 1-cell stage to maximize the chance that Cas9 cutting will occur early and therefore decrease genetic mosaicism. Embryos can be injected at later stages (2–4 cell stages), but this may possibly decrease the germline transmission rate of the modified allele.
9. Collect the eggs using a strainer and rinse them into a 10 cm Petri dish using 1x E3 media with 0.0001% methylene blue. Examine the health of the eggs under the light microscope, removing any unfertilized eggs and debris.
10. Set aside 10–15 embryos as an uninjected control in a separate, labeled Petri dish.
11. Using a transfer pipette, gently line up the eggs on the injection plate warmed to room temperature.
12. Under a dissection microscope at 2.5X magnification, inject 1 nL of the solution into the yolk sac of each embryo to inject a total of 400 pg of Cas9 protein and 200 pg of sgRNA.
    NOTE: To increase cutting if desired or necessary, increase the final concentration of Cas9 protein to 800 pg/nL and of sgRNA to 400 pg/nL in the injection solution; however, this may also increase off-target cutting and/or decrease embryo health. Cutting efficiency may also be increased by injecting directly into the cell. However, injection into the yolk sack is technically less demanding and gives sufficient cutting to produce fish with high germline transmission (>70% of offspring containing a modified allele).
13. Return the injected embryos to a properly labeled Petri dish, cover them with 1x E3 media with methylene blue, and put them in an embryo incubator set to 28 °C.
14. At 24 h post fertilization (hpf), inspect the health of the injected embryos, removing dead or abnormally developing individuals and change the media (See Figure 1). Check the rate of survival against the uninjected control.
    NOTE: When targeting a nonessential gene, less than 10% lethality is expected relative to the uninjected control. If elevated levels of lethality are observed in the guide-injected populations compared to the uninjected control, it may indicate that the targeted gene is essential for development, or off-target effects are leading to failed development. Reducing the amount of injected CRISPR-reagents may be necessary or generation of a new sgRNA with reduced off-target effects may be required.
15. Return the embryos to the incubator and continue growing the embryos to 72 hpf, changing the media daily to maintain embryo health.

Wyniki

Figure 1: Comparison of the health of 24 hpf injected embryos. A living embryo (A) developed to 24 hpf, is easily distinguished from an embryo that has aborted development (B). Embryos that resemble (B) or have drastically altered features to (A), such as spinal curvature or altered head and eye development should be removed from dish

Materiały

Name	Company	Catalog Number	Comments
RNase-free water	Any brand		Synthesis of guide RNAs. Thermo  Fisher and Qiagen both carry  suitable water.
E3 embryo media	Made in house		Media for raising embryos and making injeciton plate. Recipe: http://cshprotocols.cshlp.org/ content/2011/10/pdb.rec66449
Methylene Blue	Sigma-Aldrich	M9140	Added to 1x E3 media to prevent fungal growth on embryos.
Petri dish	Thermo Fisher	FB0875711Z	Store embryos, cast injection plate
Agarose	Denville	CA3510-8	Casting injection plate, agarose gels.
Microinection mold	Adaptive Science Tools	TU-1	To create wells to hold embryos during injection
Phenol Red	Sigma-Aldrich	P0290	Dye for visualization of injection
Mineral Oil	Sigma-Aldrich	M5904-5ML	To calibrate needle injection volume.
Transfer pipettes	Any brand		Moving embryos
Razor blade	Thermo Fisher	11295-10	Cutting injection needle, tail clipping adult fish.
Incubator			Maintaining embryos at 28.5 C.
Verticle pipette puller	David Kopf Instruments	700C	Geneate needles for injection. Additional needle pulling instructions: http:// cshprotocols.cshlp.org/ content/2006/7/pdb.prot4651.long
Capillary tubes	Sutter Instruments	BF100-58-10	Geneate needles for injection.
Microloader tips	Eppendorf	930001007	Load solution into injection needles
Microinjector	World Precision Instruments	PV 820	Injecting embryos.
Disecting microscope	Leica		Injecting embryos.
Microwave	Any brand		Casting injection plate, agarose gels.
Scale	Any brand		Casting injection plate, agarose gels.
Gloves	Any brand		For all aspects of the protocol.
N2	Any brand		To expell liquid from the capillary for embryo injection
PCR strip tubes	Any brand		For all aspects of the protocol.
200 µL tips	Any brand		For all aspects of the protocol.
10 µL tips	Any brand		For all aspects of the protocol.
Micropipettes	Any brand		For all aspects of the protocol.