Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 protein into channel catfish, Ictalurus punctatus embryos for gene editing. The development of a reliable and efficient protocol for targeted gene editing in channel catfish, is crucial to studying functional genomics. Especially after the genomic resources have been enriched by the sequencing of the channel catfish genome.

Here we present a detailed protocol for CRISPR/Cas9 genome editing in channel catfish. This protocol has been developed and validated by targeting two channel catfish immune related genes. The Toll/interleukin-1 receptor domain containing adapter molecule, or TICAM1 gene, and the rhamnose-binding lectin or RBL gene.

All of the procedures were performed at the fish genetics research unit of the EW Shell Fisheries Research Center at Auburn University in Alabama. Brood stock selection and spawning. Choose healthy channel catfish brood stock from a strain or genetic line that you would like to edit.

Channel catfish males and females should exhibit good secondary sex characteristics. Select males that have broad muscular head that is wider than the rest of their body with a well developed genital papilla. Dark color and scarring wounds from territorial fighting are also signs of sexual readiness.

Select females that have heads that are narrower than the rest of their bodies and have a soft palpable well-rounded abdomen. For accuracy and to avoid stressing the fish during initial handling, stop feeding females two to three days before examining their readiness for spawning. Fish handling should be done quickly but carefully to reduce handling stress.

All procedures that require handling of the females should be done while fish are held in spawning bags. Just before hormone injection, obtain the body weight of the females. Spawning bags with females inside can then be hung in a flowthrough tank with continuous water flow and adequate aeration.

Aseptically load the implanter that has a 14 gauge needle with 85 micrograms per kilogram of luteinizing hormone releasing hormone analog.LHRHA. Rinse the injection site and the dorsal musculature with 0.9%sterile saline solution before the injection. Insert the implanter at a 45 degree angle and release the implant.

Withdraw the needle and wipe the injection site with a cotton previously soaked in 70%ethanol. Place the spawning bag in the holding tank so the fish is completely submerged in water 15 to 20 centimeters below the water surface. Adequate aeration including five or more parts per million of dissolved oxygen, good water quality are important for ovulation of high quality eggs.

Predict the ovulation time based on water temperature using the degree hour relationship. Usually the first check for eggs is done at about 36 hours after hormone injection and then at four hour intervals. To check for ovulation gently lift the spawning bag above the water surface while you're looking for the eggs that are attached to the inside of the bag.

When you see eggs at least 10 or so this female is ovulating and ready for hand stripping. Sperm preparation. Sperm can be prepared a few hours before the expected ovulation time.

The male fish are euthanized and the testes are collected in a small weighing pan. Determine the weight of the testes. Remove any tissue and wash the testes with a 0.9%saline to remove blood if necessary.

Well developed testes are white with many villiform projections. Crush the testes and filter the sperm using a 100 micron mesh. Use 0.9%saline solution to wash the sperm from the mesh into a 50 milliliter tube.

Saline solution can be added up to 10 milliliters per gram of testes. Determine the concentration of the sperm. Check the motility and viability.

However, for these procedures this is not necessary if the testes are well developed. Sperm can then be stored at four degree C and should be used within 24 hours after preparation. Egg collection and fertilization.

Apply a very thin layer of vegetable shortening to a 20 centimeter diameter clean dry spawning pan. Place the female in an anesthetic solution containing 100 parts per million of buffered tricaine methanesulfonate with sodium bicarbonate until the fish is completely anesthetized. Carefully remove the fish from the spawning bag and dip it in 0.9%saline to wash off the anesthetic.

Dry the fish off completely using a clean towel avoiding removal of the mucus layer on the fish body surface. Apply vegetable shortening to the area around the vent including the pelvic fins to present attachment of the eggs during hand stripping. Hand strip the eggs into the greased spawning pan by applying gentle pressure to the ovaries.

Good eggs should flow freely, be golden yellow in color and have minimal or no clumps or blood clots. Avoid contact between the eggs and the water before fertilization as water can stimualte the micropyle closure. To fertilize the eggs for micro injection, transfer to 200-300 eggs to greased spawning pan.

Add one to two milliliters of the sperm solution to the eggs and mix gently. Add fresh water to the eggs to activate the sperm and eggs. Enough water should be added to slightly cover the eggs.

Gently swirl the eggs for 30 seconds. Fertilization should occur in one to two minutes. It's important to fertilize the eggs in a single layer.

Catfish eggs adhere to each other making it difficult to micro inject multiple layers of embryos. Add more fresh water to the fertilized eggs and allow the eggs to harden for 10 to 15 minutes. The remaining unfertilized eggs can be covered by wet towel to prevent drying and can be fertilized over a few hours.

Fertilization can be done in a staggered manner. One batch of two to 300 eggs can be fertilized every 30 to 60 minutes to ensure the continuous supply of embryos at the one Cell stage for the micro injection and control non-injected embryos. Needle pulling and loading.

Pull a one millimeter OD borosilicate glass capillary into two needles. Store the needles in a paper box with a piece of sponge in which several incisions have been made. To avoid breaking the needle, the diameter of the sponge should be smaller than the length of the needle stem.

The needle tip can then be opened by breaking a small piece of the needle in the finest region using a sharp object. Alternatively the needle can be opened by pinching off the thinnest region of the tip with a pair of forceps under the microscope. Prepare the injection solution by mixing guide RNAs with Cas9 protein.

Phenol red can be added to color the guide RNA Cas9 protein mix, with phenol red constituting up to one third of the total volume. The mixture is then incubated for 10 minutes on ice before use. With a micro loader, load 5-10 microliters of the guide RNA Cas9 mixture into the injection needle.

By inserting the micro loader into the needle stem and expelling the mixture slowly while retracting the micro loader tip. Avoid trapping air bubbles inside. Attach the needle to the micro pipette holder and ensure tight connection.

And then attach the holder to the micro manipulator. Ensure free and stable movement. Apply pressure for micro injection by opening the nitrogen cylinder and adjusting the pressure regulator.

The volume of injection can be affected by the pressure, the diameter of the needle aperture and the duration of the injection. To determine the volume you wish to inject, inject into a drop of mineral oil placed on a hemocytometer. If needed, the pressure and the needle diameter can be adjusted to increase or decrease the volume of the injection.

Inject multiple times in the mineral oil to ensure consistent volume. Micro injection of catfish embryos. Apply a very thin layer of vegetable shortening to 100 millimeter clean Petri dish.

Transfer 50 to 100 eggs from the fertilization pan to the Petri dish and cover them with Holtfreter's solution. The eggs should be aligned against each other in a single layer. This alignment should hold the eggs in place during the micro injection process.

Place the Petri dish with the eggs on the stage of the microscope and hold it with one hand. Lower the needle with the other hand until it pierces the chorion and yolk in a single smooth movement. The tip of the needle should be as close as possible to the blastodisc before delivering the injection material.

Depress the pedal and deliver the injection material into the yolk. If the blastodisc is not clearly visible, then the injection material can be spread into different locations throughout the yolk. To do that, the needle is inserted to the far end of the yolk.

Then depress the pedal and withdraw the needle simultaneously. This spreads the injection material at different locations throughout the yolk. 50 nanoliters containing 2.5 nanograms of guide RNA and 7.5 nanograms of Cas9 protein can be injected into the yolk without any problems.

Retract the needle smoothly as to not damage the egg. Then move the Petri dish to inject another egg with the same procedure. Avoid movement of the Petri dish during the injection process as this may damage the egg or break the needle.

Remove the eggs that are ruptured or damaged due to micro injection. Injection can be started 15 to 20 minutes after fertilization, and continue as long as the embryos are still in the one cell stage. Injected embryos are placed back in Holtfreter's solution with 10 PPM of doxycycline and continuous aeration for six to seven days until hatch.

The solution is changed daily and dead embryos are removed.Results. This figure shows the CRISPR/Cas9 induced mutations in the TICAM1 gene of channel catfish. The blue sequence represents the protospacer adjacent motif or PAM, while the green sequence represents the target for the guided RNA.

A double strand break induced by Cas9 protein was expected to occur at the site of the two red triangles. Deletion mutations are represented by a dash line, in which each dash corresponds to a nucleotide that has been deleted. Red sequences are insertions while purple represents substitution mutation.

Fractions of 78 represent the number of mutated alleles in 78 sequencing reactions. For example, 3/78 means an allele was detected in three sequencing reactions from a total of 78 reactions. The mutation rate was 79.2%in 24 individuals analyzed.

Mutations resulted in removal of a few too many amino acids from the protein, changing the downstream reading frame and premature termination of translation. In most mutations, more than 80%of TICAM1 predicted protein sequence was truncated due to premature stop codon. This figure shows CRISPR/Cas9 induced mutations in the RBL gene of channel catfish.

Exonic sequences are upper case while intronic sequences are lower case letters. Blue sequences represent the protospacer adjacent motif while the green sequence represents the target for guided RNA. A double strand break induced by Cas9 protein was expected to occur at the site of the two red triangles.

Deletion mutations are represented by a dash line in which each dash corresponds to a nucleotide that has been deleted. Red sequences are insertions. Fractions of 40 represent the number of mutated alleles in 40 sequencing reactions.

For example, 2/40 means that an allele was detected in two sequencing reactions from a total of 40 reactions. The mutation rate was 87.5%in 40 individuals analyzed. Deletions range from five base pairs to 183 base pairs while up to 20 base pairs were inserted.

More than 70%of indels resulted in truncated protein that was 10%or less than the length of the wild type protein. In this video, we demonstrated how to micro inject CRISPR/Cas9 protein into one cell channel catfish embryos. The procedures described are simple, rapid, and efficient.

And successfully achieved gene knockout in two channel catfish genes. TICAM1 and RBL. Depending on what is being injected, the protocol can be modified to include other biologically active substances.

It can also be modified to micro inject other catfish species or other fish species that have similar egg structure and composition.