CRISPR/SpCas9 Mediated Screening of p53-Related miRNA Gene Targets
Withers, Daniel Pashang
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The p53 network plays a vital role in regulating cell growth and protecting against DNA damage. Recent research has uncovered that many microRNAs (miRNAs) are activated by p53 and assist in its regulatory functions. These miRNAs act by targeting sequences on messenger RNA (mRNA) transcripts that bare partial or complete complementarity to the seed region of the miRNA. Thus, miRNAs can affect apoptosis, cell-cycle arrest, mesenchymal-epithelial transition, and stemness in cells depending on their targets. Using CRISPR/SpCas9, the targets of p53-related miRNAs were studied, as well as their relation to cell viability by building a sgRNA library. The sequences that miRNAs interact with were edited in the 3’untranslated region (3’UTR) of mRNAs and negatively select against the cells by triggering cell death with miRNA transfection. This library provides a novel approach to miRNA network analysis by allowing for high throughput analysis via edge removal.