Identification of Novel Regulatory Genes in Acetaminophen Induced Hepatocyte Toxicity by a Genome-Wide CRISPR/Cas9 Screen
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Acetaminophen (APAP) is a commonly used analgesic responsible for over 56,000 overdose-related emergency room visits annually. A long asymptomatic period and limited treatment options result in a high rate of liver failure, generally resulting in either organ transplant or mortality. The underlying molecular mechanisms of injury are not well understood and effective therapy is limited. Identification of previously unknown genetic risk factors would provide new mechanistic insights and new therapeutic targets for APAP induced hepatocyte toxicity or liver injury. This study used a genome-wide CRISPR/Cas9 screen to evaluate genes that are protective against or cause susceptibility to APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR/Cas9 gene knockouts were treated with 15mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap with gene expression data of APAP overdosed human patients, and 3) biological interpretation including assessment of known and suspected APAP-associated genes and their therapeutic potential, predicted affected biological pathways, and functionally validated candidate genes. This screen is the first genome-wide CRISPR/Cas9 knockout screen of APAP induced hepatocyte toxicity. The top hits from this screen included numerous genes previously not linked to liver injury. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. A negative selection screen identified genes involved in fundamental processes, including NAAA, ATG2B, and MYOZ3. A positive selection screen identified numerous genes potentially involved in pathogenic processes, including LZTR1, PGM5, and EEF1D. A top essential pathway at 24 hours of APAP treatment was Regulation of Skeletal Muscle Contraction. We additionally identified 6 genes, 3 novel and 3 known, that have drug-gene interactions favorable for re-purposing existing therapies to treat APAP-induced hepatotoxicity. Collectively, this line of research has illustrated the power of a genome-wide CRISPR/Cas9 screen to systematically identify novel genes involved in APAP induced hepatocyte toxicity and to provide potential new targets to develop novel therapeutic modalities.
Table of Contents
Introduction -- Review of the literature -- Research question -- Methods -- Results and discussion part 1: CRISPR/Cas9 screen -- Results and discussion part 2: our screen in the context of other acetaminophen data sets -- Results and discussion part 3: acetaminophen-associated single nucleotide polymorphisms in the literature -- Results and discussion Part 4: validation of top candidate genes -- Conclusions and future directions -- Appendix A. Supplementary figures -- Appendix B. Supplementary tables
Ph.D. (Doctor of Philosophy)