Having knocked-out MMP-14 expression pre-translationally (morpholino) and post-translationally (chemical inhibition), our next investigation into the roles of the MMP-14 protein featured a pre-transcriptional knock-out through the use of the cutting-edge CRISPR/cas9 system. A unique feature of our model organism (Xenopus laevis) is that it has a tetraploid genome, as opposed to a diploid system in humans and other mammals. Thus, per chromosome, instead of a single pair of homologous chromosomes, X. laevis has two pairs, termed the “long form” and “small form.” As a result, it is necessary to produce two guideRNAs, one for the long and one for the short form, to be injected alongside the cas9 protein. The use of the CRISPR system, like that of the morpholino, shall be site-specific through microinjection, and assessed through in-situ hybridization with a sox10 probe. Through use of these guideRNAs and the cas9 protein, we hope to increase our understanding of MMP-14’s role in neural crest migration through a pre-transcriptional lens.
Hypothesis:
It is expected that embryos injected with short form and long form guideRNAs will feature a stunted migration and unsuccessful segregation of migratory streams of neural crest cells upon reaching stage 24. This result is expected based upon previous in-situ hybridizations of embryos treated with chemical inhibitor or morpholino. The effect, however, is expected to be smaller than either of the two other treatments given the unreliable nature of the CRISPR/cas9 system.
Experimental Design:
Synthesizing GuideDNA:
Transcription of GuideDNA to Yield GuideRNA:
Obtaining GenomicDNA Specific to the MMP-14 Gene:
Cleavage Assay and the Efficiency of the GuideRNAs:
Microinjection of GuideRNA and cas9 Protein:
Results:
Hypothesis:
It is expected that embryos injected with short form and long form guideRNAs will feature a stunted migration and unsuccessful segregation of migratory streams of neural crest cells upon reaching stage 24. This result is expected based upon previous in-situ hybridizations of embryos treated with chemical inhibitor or morpholino. The effect, however, is expected to be smaller than either of the two other treatments given the unreliable nature of the CRISPR/cas9 system.
Experimental Design:
Synthesizing GuideDNA:
Transcription of GuideDNA to Yield GuideRNA:
Obtaining GenomicDNA Specific to the MMP-14 Gene:
Cleavage Assay and the Efficiency of the GuideRNAs:
Microinjection of GuideRNA and cas9 Protein:
Results: