Dip2a Knockout Using Crispr/Cas9Technology

The research was conducted in order to understand the roles that Dip2a gene plays in an organism. Therefore, generation of Dip2a knockout mice using CRISPR/Cas9Genome Engineering was done to get transgenic mice with disrupted Dip2a gene. Here,65Kb was cleaved from Dip2a gene through CRISPR-Cas9technology whereby sgRNA was employed to target two separate sites in the gene namely first intron and3’UTR. The oligos of intron1and3’UTR were used in the construction of sgRNA expression vector. Donor vector was also constructed which was used in Embryonic Stem cells transfection together with the expression vector and also Rad51to test recombination efficiency. The transfected Embryonic Stem cells were tested for both recombination efficiency as well as deletion efficiency through PCR genotyping. Direct microinjection of the constructed DNA expression vector was done into mice zygotes which later were transferred into foster mothers which gave birth to pups. Green fluorescent protein (GFP) test was done on the pups to identify the knockouts and then PCR genotyping was done to see both deletion efficiency and identification of knockout mice. DNA sequence was done to confirm the cleavage. Western blot and Real Time PCR was to be conducted for phenotypic establishment hence identification of dip2a roles. Both PCR genotyping results at cell level and knockouts showed an amplified band of approximately600bp signifying that knockout happened thus with deletion efficiencies of4.3%in ES cells and25%in mice whereas recombination efficiency was averaged at32%in ES cells that were not transfected alongside Rad51whereas those transfected together with Rad51it was averaged at66%. GFP test, genotyping and DNA sequencing confirmed presence of knockouts in the pups. Finally, phenotypical tests are to establish roles played by Dip2a in an organism.