This protocol describes a method for quickly screening thousands of genomes using F0 CRISPR-injected zebrafish sperm. This technique is advantageous in its accessibility. Rather than expensive, specialized sequencing-based approaches, our method uses PCR restriction enzymes and gel electrophoresis to screen F0 male carriers for indels or specific genome edits.
The hardest part of this procedure is just getting the sperm quickly, but it becomes a good technique for in vitro fertilization if we work with a lot of dysmorphic fish, and so it's an easy way for us to cross fish that are giving us trouble. Begin by setting up breeding tanks for both the wild type and F0 fish and incubate the tanks overnight. After anesthetizing the male fish the next day, use a slotted spoon to transfer it onto a stack of clean paper towels.
Gently roll the fish to remove excess water. Remove any water by blotting it dry with a clean folded tissue. Using the slotted spoon, transfer the fish to the prepared sponge.
Place the fish ventral-side up and gently blot the area around the anal fins with a clean folded tissue paper. To collect the sperm, use a capillary tube to gently move the pelvic fins away from the midline, exposing the cloaca. Place the capillary tube near the cloaca.
With your fingers or a pair of filter forceps, gently squeeze the sides of the fish from the gills down. Using capillary action, collect the sperm in the tube and place it in a PCR tube containing 40 microliters of 50 millimolar sodium hydroxide solution, then expel the sample into the tube. After spinning the sperm samples in a mini centrifuge, place the PCR tubes into a thermal cycler.
Run the cycler by heating the samples for 40 minutes at 95 degrees Celsius followed by cooling at 25 degrees Celsius. After removing the samples from the cycler, neutralize the pH by adding 10 microliters of one molar tris hydrochloride of pH eight. Mix by aspirating the suspension and then centrifuge the samples.
Set the thermal cycler to 95 degrees Celsius for preheating. And in the meantime, prepare 25 microliters of the PCR reaction mixture for each sperm sample. Mix well by pipetting up and down, then spin the samples.
Place the samples in the preheated thermal cycler and set the cycle. To obtain a gel solution, microwave a 4%agarose gel solution prepared by mixing agarose powder, gel stain, and TBE buffer. Pour the gel solution into a gel casting frame and insert a comb on one side.
After gel solidification, carefully remove the comb. Place the gel in an electrophoresis rig such that the wells are closest to the negative electrode. Pour the TBE running buffer into the rig until the wells are fully submerged.
Begin to load the gel by pipetting five microliters of DNA ladder into the first well. Load the remaining wells with 5 to 10 microliters of the PCR product. Run the gel for 1 hour at 150 volts to ensure adequate separation of the amplicons.
Use the gel imager to view the DNA bands. p2ry12 locus analysis showed that the wild-type amplicon displayed a single band of 250 base pair length while the F0-injected male amplicons containing indels ran as multiple bands. dnah10 locus analysis showed that wild-type amplicon run as a single 400 base pair long band.
F0-injected male amplicons containing indels ran as multiple bands or as a single band with decreased gel mobility. The F0-injected male number one had an additional band in the digested product, which was absent in the PCR product, rendering it the best founder candidate for mutant knock-in line establishment. If a male fish does not produce sperm when squeezed, it is best to rest the fish for a week and try again instead of squeezing them too hard and risk hurting them.
Once the FC or male founder is outcross, the alleles need to be sequence verified in the F1 progeny. Sequencing the F1 amplicons directly and performing heterozygous allele analysis is an easy way to do this.
