Improving the efficiency of CRISPR-Cas9:  The expression & purification of Cas9

Undergraduate Honors Thesis

Citations

Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/g732d951z

Abstract

p53 is a transcription factor that plays a significant role in cell cycle regulation, senescence, and apoptosis. The p53 protein has been widely studied for its link to human cancer, and is estimated to be mutated in ~50% of all human cancers. This project focused on generating a p53-null A549 cell line using the genome editing technique CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9. CRISPR-Cas9 was employed to generate a simultaneous knock-in/knockout of mCherry and WTp53, respectively. The mCherry gene was inserted at the WTp53 endogenous translational start site (TSS), thereby rendering WTp53 inactive and mCherry “active” whenever WTp53 would normally be expressed. After the initial transfection yielded low levels of mCherry fluorescence, focus shifted to next-generation CRISPR techniques that would increase the p53 knockout efficiency. One of these techniques included expressing and purifying the Cas9 protein in order to directly introduce the ribonucleoprotein (RNP) complex to target cells via electroporation. Cas9 was overexpressed in Rosetta 2 (DE3) E. Coli cells and was purified through a Ni-NTA affinity column followed by a Heparin Ion-Exchange column. The Cas9 purification process has been optimized for future uses, and the assembly and electroporation of Cas9 RNPs will likely increase efficiency for any upcoming genome-editing projects within the Taatjes lab.

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