Endogenous epitope tagging of heat shock protein 70 isoform Hsc70 using CRISPR/Cas9

Abstract

Heat shock protein 70 (Hsp70) is an evolutionarily well-conserved molecular chaperone involved in several cellular processes such as folding of proteins, modulating protein-protein interactions, and transport of proteins across the membrane. Binding partners of Hsp70 (known as "clients") are identified on an individual basis as researchers discover their particular protein of interest binds to Hsp70. A full complement of Hsp70 interactors under multiple stress conditions remains to be determined. A promising approach to characterizing the Hsp70 "interactome" is the use of protein epitope tagging and then affinity purification followed by mass spectrometry (AP-MS/MS). AP-MS analysis is a widely used method to decipher protein-protein interaction networks and identifying protein functions. Conventionally, the proteins are overexpressed ectopically which interferes with protein complex stoichiometry, skewing AP-MS/MS data. In an attempt to solve this issue, we used CRISPR/Cas9-mediated gene editing to integrate a tandem-affinity (TAP) epitope tag into the genomic locus of HSC70. This system offers several benefits over existing expression systems including native expression, no requirement for selection, and homogeneity between cells. This cell line, freely available to chaperone researchers, will aid in small and large-scale protein interaction studies as well as the study of biochemical activities and structure-function relationships of the Hsc70 protein.

Dynamic remodeling of the interactomes of Nematostella vectensis Hsp70 isoforms under heat shock

“…Experiments on Ssa isoforms in yeast have hinted that there may be isoform specific function and client specificity [3,[40][41][42][43][44]. The advent of genomic epitope tagging of chaperones via CRISPR has made it possible to study chaperones complexes in their native stoichiometry but such approaches remain in their infancy in non-model organisms like Nematostella vectensis [45,46].…”

CRISPR/Cascade 9-Mediated Genome Editing-Challenges and Opportunities

“…This vector delivery approach is efficient for gene editing, reaching 85% efficiency (Xiong et al, 2017 ). Finally, a recently described CRISPR-mediated epitope tagging approach holds promises for efficient gene knock-in (Nitika and Truman, 2017 ). So far, NHEJ-mediated repair of CRISPR/Cascade 9-mediated insertion of large DNA fragments has not been investigated in humans, and effective knock-in in human embryonic stem cell (ESCs) still constitutes a challenge.…”

Chemogenomic screening Identifies the Hsp70 Co-chaperone HDJ2 as a Hub for Anticancer Drug Resistance

“…Several Hsp70 inhibitors have failed in clinical trials due to their toxicity. More recently, alternative strategies have focused on sensitizing cells to anticancer agents by either manipulating post-translational modification of chaperones or their interaction with specific co-chaperones [4, 9–19]. HDJ2 (mammalian homologue of yeast Ydj1) is an interesting possible anticancer target as a key mediator of Hsp70 function that appears to regulate specific features of tumorigenesis [13, 20, 21].…”