Applying CRISPR Cas9 to the Hair Follicle Neogenisis Implicated Wnt10B Gene

Overview

The purpose of this project is to design and test synthetic guide RNA (sgRNA) used in CRISPR/Cas9 techniques to cut Wnt10B target regions for inserting an enhancer. The Wnt10B gene is part of the Wnt signaling pathways which are a group of signal transduction pathways that start with proteins and end with surface cell receptors. The Wnt10B gene in humans is 96% similar to mice. Researchers have found that Wnt10B is implicated in hair follicle neogenesis after wound healing in adult mice. Hair follicle neogenesis is a process previously thought to only be possible during embryonic development. Inserting an enhancer upstream of the Wnt10B gene is the mouse genome would allow overexpression of said gene during the wound healing process in order to observe its phenotypical expressions. After designing the sgRNA primers, several processes, including transfection, flow cytometry, gel electrophoresis, ligation and polymerase chain reaction (PCR) and Sanger sequencing were used to test the product. Sequencing showed that the sgRNA were able to accurately cut the desired regions, about 3-4 base pairs away from the PAM sequence, however, the overall effectiveness of the product was not confirmed due to the low quality of the DNA samples. In conclusion, the sgRNA showed promise, but the overall effectiveness of the products were inconclusive. Therefore further studies must be conducted to test the sgRNA as well as the nature of the Wnt10B gene which may affect efficiency of the sgRNA.

Competition history

• AJAS 2019 Category not listed

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