This protocol allows researchers to build a number of previously unavailable zebrafish models of disease-relevant single-nucleotide variants. PAM limitations are major obstacle to creating accurate animal models, and zSpRY-ABE8e enables efficient adenine to guanine base conversion with low intermutation and relaxed PAN restriction. This tool can be used to construct accurate zebrafish models for studying the pathogenic characteristics disease mechanisms related to single-nucleotide marines, as well as drug screening for this disease.
To begin, set up the micropipette puller system and preheat for at least 30 minutes. To pull the glass capillaries, select a program, set the ramp test value as heat, 50 as pull, the velocity at 100, time as 50, and pressure as 30. Next, install the capillary tube ensuring it is in the groove.
Press pull start/stop"to run the program. To preserve the pulled capillaries, insert them into foam containing gaps keeping the needle suspended. For experimentation, use zSpRY-ABE8e mRNA described in the manuscript, and easy edit sgRNA modified with 2'O-methyl phosphorothioate at both ends.
For the gRNA design, preferentially select NRN as the PAM sequence, as zSpRY-ABE8e shows a higher preference for NRN PAM than NYN PAM. Enter the target adenine nucleotide is in the highly active editing window from the third to the ninth nucleotide along the protospacer. Set up breeding tanks the night before the injection.
Insert a divider, placing two female and one male zebrafish in each tank. Place a lid over each tank. The next morning, using RNase-free tips, tubes, and gloves, mix the thawed mRNA and EEgRNA on ice to a final concentration of 400 and 200 nanograms per microliter, respectively.
To configure the pneumatic microinjector, first, close the partial pressure valve of the air pump. Then, open the main valve, unscrew the gas cylinder, and adjust the pressure of the partial pressure valve to 0.2 Megapascals. Now, the turn on the microinjector and set the mode to timer.
Adjust the parameter pressure valve to 20 pounds per square inch and the timer value to 0.040 seconds. Using fine forceps, angularly break the needles of the pulled glass capillaries under a stereo microscope. Using a microloader pipette tip, add the mRNA/gRNA mixture to the tip of a glass capillary and attach the needle to the micromanipulator.
Configure the pressure and timer value to produce two nanoliters mixture per injection using a microcap. After breeding the fish for 10 to 15 minutes, collect the eggs in a strainer. Examine the cell stage and egg quality under the stereo microscope.
Select single-celled eggs for injection to improve the editing efficiency. Line the eggs up on a 1.5%agarose injection plate and inject two nanoliters of the RNA mixture into the cell of the embryos using a microscope. Collect the eggs in Petri dishes with E3 Buffer and incubate them at 28 degrees Celsius.
Remove the dead eggs and change the culture buffer every 12 hours for 48 hours post-fertilization. After 48 hours post-fertilization, collect three pools of six randomly selected injected embryos into a PCR tube. Once six randomly selected control embryos are collected, use a pipette to aspirate the residual E3 Buffer.
Add 40 microliters of 50 millimolar sodium hydroxide to the embryos. Place the tubes in a metal bath at 95 degrees Celsius for 20 minutes, and then vortex for 15 seconds. Add four microliters of one molar Tris hydrochloride at pH eight into each tube.
Centrifuge the tubes at 2000g for 10 seconds at room temperature. Transfer the supernatant into a new PCR tube. Remove one microliter of the supernatant from the PCR tube and use it for Sanger sequencing.
Then store the remaining supernatant at 20 degrees Celsius. To analyze the Sanger sequencing data, begin by uploading the sequencing file into the upload ab1 File"box. Enter the 20 base pair gRNA sequence in the 5'to 3'orientation into the enter gRNA"sequence box.
Enter the corresponding base position into the 5'start and 3'ends boxes. Obtain the base editing efficiency by clicking on predictive editing"and checking the quality of the sequencing data. A diamond blackfan anemia zebrafish model was constructed by converting the adenine of the start codon of Tsr2 to guanine.
The EditR analysis of sequence data demonstrated an AG overlap at the adenine base of the Tsr2 start codon. The phenotype of the two day-old Tsr2 embryos showed smaller eyes compared to the control embryos. A swollen pericardium was also observed in the mutant embryos.
In order to achieve efficient base editing, please strictly follow the previously mentioned guideline design principle, which is a prerequisite for the success of the experiment. Accurate models are essential for the study of single-nucleotide variant-related diseases. The development of the SprY-ABE8e provides an effective tool for accurate modeling, which helps to promote the research of disease mechanisms and treatments.
