CRISPR/Cas9 gene editing in weakly electric fish, particularly in a comparative context, will enable researchers to test hypotheses about how specific genes and genetic variance contribute to phenotypic variation. CRISPR/Cas9 is an efficient method for generating mutant F-zero embryos. This protocol allows researchers to conduct CRISPR-Cas9 manipulations in two convergently evolved species of weakly electric fish.
The key to using this approach with some modifications in other electric fish or other fish species is the availability of transcriptomic or genomic resources to make sgRNAs. Performing the microinjections and visualizing the cells in a 3D location within a 2D plane can be difficult. Obtain a small zebrafish colony for eggs to practice general microinjection techniques.
Demonstrating the procedure with Savvas Constantinou will be Jared Thompson, a technician from my laboratory. After housing the fish species of interest under the appropriate breeding conditions, identify female fish that appear gravid. In B.gauderio, the female will have swollen gonads just caudal to the vent.
In B.brachyistius females will have swollen bellies and appear deep-bodied. After anesthesia administration, add spawning agent to four times the volume of DPBS in a PCR tube with thorough mixing. The solution will become cloudy.
Use a pipette to measure out the calculated dose and dispense the diluted agent unto a piece of thermoplastic before drawing the solution into a precision glass syringe equipped with a 28-gauge 19 to 25-milliliter length beveled needle. Inject the solution into the dorsal trunk muscle at a smooth rate and let the needle sit for two to four seconds before removal. Then, immediately place fish into fresh system water for recovery.
For B.gauderio sperm collection, after 24 hours, select a large male fish and as quickly as possible after sedation, dry the fish thoroughly, especially around the head and Vent. Place the dried fish ventral side up, anterior to the left in appropriate holder, covered in an MS222 soaked damp paper towel with the head as parallel with the table as possible. Immediately apply pressure in the caudal to rostral direction with light squeezing immediately over the gonads toward the vent and using a micropipetter with a tip, carefully collect the sperm as it is squeezed from the male in 50-microliter increments.
Add aliquot of sperm directly onto 500-microliter volumes of sperm extender solution on ice. Immediately after the collection, place fish into slime coat protection product-treated fresh system water. For B.gauderio egg collection, after drying an a anesthetized B.gauderio female fish, immediately place the fish on its side with the head facing the nondominant hand and apply pressure in the caudal to rostral direction with light squeezing medially over the gonads toward the vent.
Using a polytetrafluoroethylene tool, carefully collect the eggs as they are squeezed from the cloaca and quickly place the eggs in a small covered Petri dish. If working alone after squeezing, touch the egg mass to the base of the small Petri dish as they are expelled from the female. Immediately after collecting the eggs, place the fish in slime coat protection product-treated fresh system water.
For B.gauderio in vitro fertilization, add 100 microliters of the well mixed sperm SES solution directly over the egg mass, followed by one milliliter of fresh system water. Mix the gametes suspension thoroughly for 30 to 60 seconds before adding an additional one to two milliliters of 0.22-micrometer pore filtered system water to the cells. Label the dish with the in vitro fertilization time and place the dish into a 29-degree Celsius incubator, setting a 50-minute timer to check the progress of the development.
During the fertilization period, use a microloader pipette tip to backload two microliters of the injection solution of interest into a microinjection needle and expel the liquid as close to the tip as possible. When a single cell has formed at the animal pole of at least one cell, transfer the dish under a dissecting microscope and use a pair of fine forceps to break the needle tip at a beveled angle toward where the taper of the needle begins to demonstrate rigidity. The bore of the needle should remain as small as possible while maintaining a rigid taper.
Identify the developing zygotes under the microscope and use a plastic transfer pipette with a cut tip to place 10 to 20 eggs in as little water as possible onto the edge of the glass microscope slide set in the inverted top of the Petri dish. Using a delicate task wipe, gently press the slide to remove the excess water to allow the eggs to adhere firmly to the edge of the slide. Water will be wicked under the slide and pull eggs against the edge.
There should be just enough water to keep the eggs moist while maintaining little standing moisture to avoid egg movement during injection. Align the eggs against the slide vertically, perpendicular to the approaching needle, and use a micromanipulator to position the needle against the chorion. Then, holding the needle at a roughly 45-degree angle, insert the tip first into the chorion and then, into the single cell, injecting through the yolk if possible.
Remove any broken eggs during the injection with fine forceps. When all of the eggs has been injected, use a squirt bottle of 0.22-micrometer pore filtered system water to gently flush the eggs from the injection stage into a new 100-millimeter diameter Petri dish. Following a successful short guide RNAs selection and synthesis, in vitro cleavage can be tested for selection for single cell microinjections.
After PCR clean up and cloning, in this representative experiment, CRISPR-Cas9 induced mutations were identified in individual B.gauderio and B.brachyistius clones with strong electric organ discharge amplitude reduction, whereas uninjected controls displayed only referenced genotypes. Visualization of the electric organ discharge amplitude between confirmed CRISPR mutants and agent size matched uninjected controls demonstrated that both SCN-4 AA mutant B.brachyistius and B.gauderio embryos had significantly lower electric organ discharge amplitudes than controls. When performing the micro injections, move as quickly and deliberately as possible to maximize the number of single-cell injected embryos that survive.
Do not waste time with difficult eggs. Once successful mutants have been identified, traditional breeding methods can create stable mutant lines, which eliminate CRSPR associated mosaicism concerns. This protocol will allow researchers to conduct comparative genomic studies with functional validation in two convergently evolved species of weakly electric fish.
