CRISPR-Cas9 Transfection Optimization and Use in a Forward Genetic Screen to Identify Telomere Length Maintenance Genes

• Main Author: Phillips, Kelsey
• Format: Others
• Published: BYU ScholarsArchive 2018
• Subjects: CRISPR-Cas9; telomere; telomerase; forward genetic screen; ZNF827
• Online Access: https://scholarsarchive.byu.edu/etd/7357; https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=8357&context=etd

Mutations in the telomere length maintenance pathway can lead to a spectrum of diseases called telomere syndromes, however, the pathway is not fully understood and there may still be unknown components. We designed a forward genetic screen to identify new genes involved in telomere length maintenance. Of the top ranked genes, ZNF827, a zinc finger protein, is the most promising candidate gene. The possible discovery of a new component involved in telomere length maintenance increases our understanding of the pathway and opens new avenues of research. Recent advances in molecular biology techniques, such as the use of RNA-guided nuclease CRISPR associated protein 9 (Cas9), have made screens like this possible. Cas9 is a nuclease that uses a guide RNA(gRNA) to direct its endonuclease activity. The use of Cas9 has revolutionized the field of genome engineering, providing scientists with more efficient methods to knockout and modify genomes. We sought to optimize CRISPR-Cas9 genome editing to make it as widely accessible as possible. We compared plasmid, ribonucleoprotein (RNP), and RNA only lipid-mediated transfection of CRISPR-Cas9 into cell lines using a novel reporter system to measure genome editing efficiency. All methods were successful to some extent, however, RNP lipofection was the most efficient and has many advantages over other methods. We also found that short homology arms of 30-35bp on donor templates was able to mediate site specific editing. These methods should broaden the accessibility of CRISPR-Cas9 genome editing.