Early development is dependent on maternally-inherited products, and the role of many of these products is currently unknown. Herein, we described a protocol that uses CRISPR-Cas9 to identify maternal-effect phenotypes in a single generation.
The role of many maternally-expressed genes during early development is currently unknown. This maternal CRISPR technique allows for the rapid identification of maternal effect genes and their role in development. Multiplexing guide RNAs to a single gene, allows researchers to identify novel maternal effect phenotypes in a single generation.
This research is beneficial to understand the function of mRNA transcripts in the gametes, which are necessary for early embryogenesis. The key to this technique is to micro inject the embryos early in the one cell stage, and check to see of the majority of the guide RNAs can make somatic mutations in the injected embryos. To create a guide RNA template for each gene specific oligonucleotide, analyte to the constant oligonucleotide and fill in the overhangs with T4 DNA polymerase.
After the four guide RNA templates are assembled, purify and concentrate them together using a DNA cleanup and concentrator kit. Synthesize the sgRNA mixture from the pooled guy RNA template, using an in-vitro T7 transcription kit, and perform the in-vitro transcription as described in the manuscript. To verify the integrity of the sgRNA, cast a 1%agarose 0.5 micrograms per milliliter, ethidium bromide Tris-Borate-EDTA gel.
Place the solidified gel in Tris-Borate-EDTA running buffer. Mix one microliter sgRNA mixture, and one microliter RNA gel loading buffer. Load this sample in the gel and run the gel at 100 volts for five minutes.
Visualize the bands using ultraviolet light. Set up wild type crosses and zebrafish mating boxes. Keep the male and female fish in the same tank, but separate them using a mating box divider, or as shown here, by placing the female inside an egg laying insert.
After the injection cocktail is assembled, allow the male and female to mate by removing the mating box divider, or as shown here, by placing the male in the same egg laying insert as the female. To synchronize the embryos, collect them after 10 minutes using a plastic strainer, and rinse them into a Petri dish using 1XE3 media. Remove 10 to 15 embryos and place them into a separate Petri dish to be kept as un-injected controls.
Transfer the remaining embryos into the injection plate wells. Inject one nanoliter protein sgRNA solution, into the developing blasto disc of a one cell embryo. Use forceps to break the tip of the clogged needle and recalibrate the needle to eject a one nanoliter bolus.
The next day after injection, collect six healthy injected embryos and two control embryos from the un-injected plate. Place each embryo individually into a single well of a PCR strip tube and label the top of the tubes. Design unique screening primers for each guide site to amplify a 100 to 110 base paired DNA fragment that includes the CRISPR-Cas9 target site.
If possible, place the target site in the middle of the amplified fragment, allowing for the identification of larger deletions. For each of the four guide target sites, set up eight 25 microliters PCR reactions using five microliters of the prepared single embryogenomic DNA and 20 microliters of the PCR mix, and guide specific screening primers mixture, to identify somatic mutations in the target site. Cast a 2.5%agarose, 0.5 micrograms per milliliter ethidium bromide Tris-Borate-EDTA gel using combs that create approximately 0.625 centimeter wide wells.
Then place the solidified gel into the electrophoresis chamber containing Tris-Borate-EDTA running buffer. Add five microliters of 6X loading dye to the PCR product. Load 25 microliters of this mixture into the gel, ensuring that the injected and control samples are running on the same gel row.
After all the samples are loaded, add five microliters of ethidium bromide per one liter of Tris-Borate-EDTA running buffer, to the positive end of the gel box. Run the gel at 120 volts until the DNA bans resolve or the DNA has approached the end of the lane. To identify the maternal effect phenotypes and maternal CRISPR's embryos, set up the F0 injected females against wild type males and control wild type crosses in standard zebra fish mating boxes during the afternoon before the experiment.
Place the male and female fish in the same tank, but separate them with a mating box divider, or place the female inside an egg laying insert. On the morning of the experiment, allow the male and female to start mating by removing the mating box divider, or placing the male in the same egg laying insert as the female. Collect the embryos every 10 minutes by moving the egg laying insert into a new mating tank bottom that contains fresh system water, and label the tank with a tag identifying the individual F0 female.
Take the old mating tank and pour the water through a T-strainer to collect the embryos from one individual 10 minutes clutch. Under a dissecting microscope with a transcendent light source, observe the embryos undergoing development every hour, for the first six to eight hours, and daily for the next five days. Move the potential maternal CRISPR embryos to a Petri dish that contains 1XE3 media, and assay for morphological phenotype at 24 hours post fertilization, and viability, at five days post fertilization.
The afternoon before the experiment, set up mating pairs of F0 females by keeping the wild type males physically separated from the females, using a mating box divider or placing the female in the egg laying insert. On the morning of the experiment, remove the physical partition to initiate the mating. At the first sign of egg laying, interrupt breeding by separating the male and F0 females, and keep each separated F0 female in individual mating boxes.
After in-vitro fertilization, allow the haploid embryos to develop until the maternal CRISPR phenotype is observed, and place those embryos into a different Petri dish. Once the maternal CRISPR haploid embryos have been identified, allow them to develop for at least six hours post fertilization. To extract the genomic DNA from at least 10 maternal CRISPR haploid embryos, place a single haploid embryo into an individual well of a PCR strip tube, remove excess E3 media from the well, and add 50 microliters of 50 millimolar sodium hydroxide.
Incubate the embryos for 20 minutes at 95 degrees Celsius, and cool them down to four degree Celsius. Then add five microliters of one molar Tris-hydrochloric acid with pH 7.5, and vortex for five to 10 seconds. To identify which guide sites contain indels, design sequencing primers to amplify a DNA fragment including all four CRISPR/Cas9 target sites.
Set up two 25 microliters of PCR reactions per embryo, using five microliters of the prepared genomic DNA, and the sequencing primers. After the PCR is finished, purify and concentrate the two samples using a DNA cleanup and concentrator kit, and submit the DNA fragment to Sanger sequencing, using the forward and reverse sequencing primers. Guide RNAs should all be clustered together with minimal to no overlapping regions between guide RNAs during maternal CRISPR's generation.
If indels were created, a smear should be observed in the injected samples on an agarose gel, but not in the un-injected control. The maternal CRISPR method can be used to phenocopy known maternal effect mutations, such as motley, tmi, and aura. To identify the genetic lesions that contribute to the maternal CRISPR-phenotype, UV treated sperm and in-vitro fertilization are combined to create maternal CRISPR haploids.
The creation of a haploid allows Sanger sequencing of the maternal allele, and identification of maternal CRISPR indels. When identifying an unmaternal effect phenotype, is essential that you are able to observe it in multiple F0 females, because it increases the odds that the phenotype is caused by loss of function, not off target or non-specific effects. After a maternal effect phenotype has been identified, the maternal CRISPR embryos can be used to examine the effects on various aspects of early embryogenesis, such as the sinus skeleton or DNA segregation.
This allows researchers to understand the molecular cause of the phenotype. This method directly tests the function of unknown maternally expressed genes in a single generation, expediting our understanding of processes, acting during early embryogenesis.
