EDEM3 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed for functional investigation of endoplasmic reticulum-associated degradation (ERAD) and glycoprotein quality control. This pool of HAP1 cells harbors CRISPR/Cas9-mediated gene disruption within the EDEM3 locus, generating a loss-of-function model that recapitulates ablation of the encoded mannosidase. Because the pool contains a heterogeneous mixture of editing events, it enables robust screening applications and population-level phenotypic analyses without requiring single-cell cloning. The polyclonal format is particularly well-suited for pooled functional genomic assays and assessing the overall impact of EDEM3 deficiency on cellular homeostasis.
The host cell line, HAP1, is a near-haploid adherent cell line derived from the KBM-7 chronic myeloid leukemia (CML) background. Its near-haploid karyotype facilitates straightforward gene targeting and simplifies interpretation of genotype?Cphenotype relationships, as each cell typically carries a single functional allele for most genes. Originating from a hematopoietic malignancy, HAP1 cells retain cancer-relevant signaling networks and are widely employed in cancer biology, drug sensitivity profiling, and CRISPR-based functional screens. The hematopoietic context makes this model particularly relevant for studying proteostasis pathways that support leukemic cell survival.
EDEM3 encodes an ER-resident ??-1,2-mannosidase that trims specific mannose residues from asparagine-linked (N-linked) glycans on misfolded glycoproteins. This trimming generates a degradation signal recognized by the ERAD lectins OS-9 and XTP3-B, which deliver substrates to the HRD1 ubiquitin ligase complex. Within this complex, SEL1L acts as an adaptor, facilitating interaction with the p97/VCP ATPase, which drives retrotranslocation of substrates from the ER to the cytosol for proteasomal degradation. EDEM3 expression is induced by ER stress through the IRE1/XBP1 and ATF6 arms of the unfolded protein response (UPR), placing it downstream of these sensors and upstream of the HRD1-SEL1L-p97-proteasome axis. As a result, EDEM3 functions as a critical gatekeeper in the ERAD branch of cellular protein quality control.
In the HAP1 CML background, loss of EDEM3 disrupts the normal disposal of terminally misfolded glycoproteins, potentially leading to accumulation of ERAD substrates and chronic ER stress. This genetic context allows investigation of how hematopoietic cancer cells cope with proteotoxic pressure and may reveal dependencies on the HRD1 complex or alternative degradation routes. The near-haploid nature of HAP1 further simplifies detection of downstream functional consequences, such as changes in viability, UPR activation, or N-glycan profiles, making it a powerful system for probing ER-stress-associated vulnerabilities in cancer.
EDEM3 Knockout HAP1 Polyclonal Cells serve as a versatile platform for dissecting ERAD and UPR mechanisms and for screening modulators of ER stress. Researchers can monitor canonical UPR markers by western blotting or RT-qPCR, assess transcriptome-wide changes by RNA-seq, and visualize substrate accumulation using immunofluorescence or flow cytometry. The XBP1 splicing reporter assay offers a sensitive readout of IRE1 activity, while proteasomal activity assays directly measure degradation capacity. This knockout model is therefore applicable to studies of protein misfolding diseases, congenital disorders of glycosylation, and cancer cell adaptation to proteotoxic stress, as well as to drug-discovery programs targeting the ERAD pathway. For further information, please contact Ascent Research.
