En'Cas'ing The Stress: Engineering a Human Cell Line Knockout of Heat Shock Response Genes

The Heat Shock Response (HSR) is an evolutionarily conserved response to high temperatures and other stresses that controls adaptive proteostasis, and is primarily regulated by the factor, Heat shock transcription factor 1 (HSF1).  In mammalian cells, HSF1 is converted from an inactive monomeric form to an active trimer in response to heat stress. A ribonucleoprotein complex comprising of eukaryotic translation elongation factor eEF1A1, and a long noncoding RNA HSR1 are the key components of HSF1 activation. Once activated, HSF1 is recruited to Heat Shock Protein (HSP) promoter regions, upregulating chaperone activity in the cell. Along with mediating initiation of HSR, eEF1A1 is also a vital component of protein synthesis machinery. Interestingly, another isoform of eEF1A, called eEF1A2, is expressed in some specialized terminally differentiated cells of skeletal muscle, heart, pancreatic islets and motor neurons, all of which are prone to protein aggregation. The two isoforms are 92% identical and are reciprocally regulated. To better understand the role of eEF1A1 and HSF1 proteins in humans, we use a CRISPR-Cas9 nickase system to knockout HSF1 and eEF1A1 in a human cell line. We showed that the hTERT-immortalized, normal diploid foreskin fibroblast cell line, BJ-5ta, produces both eEF1A isoforms. This will allow us to perform eEF1A1 knockout in these cells. We hypothesize that HSF1 knockout cell line will survive under normal conditions but express very low thermotolerance. Conjointly, we hypothesize that the elimination of eEF1A1 may be compensated by the upregulation of eEF1A2. If viable, the eEF1A1 knockout cell line will be used for screening mutants of eEF1A2 restoring activation of HSR. Both HSF1 and eEF1A1 knockout lines will also be used for future studies to improve upon the current model of the HSR pathway and potentially reveal therapeutic targets for diseases like ALS, Alzheimer's disease, Parkinson's disease, type 2 diabetes, and amyloidosis.