ArticleBiochemistry and biophysics reports2022
CRISPR/Cas9 disruption of EpCAM Exon 2 results in cell-surface expression of a truncated protein targeted by an EpCAM specific T cell engager.
Article in Biochemistry and biophysics reports, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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4 citing papers in PubMed, 5 citations in OpenAlex.
- Engineering novel CRISPRi repressors for highly efficient mammalian gene regulation.Genome biology · 2025Article
- Construction of EpCAM overexpression and knockdown vectors and their implications in colorectal cancer research.Frontiers in genome editing · 2025Article
- Escaping from CRISPR-Cas-mediated knockout: the facts, mechanisms, and applications.Cellular & molecular biology letters · 2024Review
- A CRISPR-del-based pipeline for complete gene knockout in human diploid cells.Journal of cell science · 2023Article
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4 authors at 1 institution in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
CRISPR/Cas9 gene-editing technology allows researchers to study protein function by specifically introducing double-stranded breaks in the gene of interest then analyze its subsequent loss in sensitive biological assays. To help characterize one of a series of highly potent, conditionally active, T cell engaging bispecific molecules called COBRA™, the human EpCAM gene was disrupted in HT29 cells using CRISPR/Cas9 and guide RNA targeting its Exon 2. Although a commercially available antibody indicated loss of cell-surface expression, the EpCAM targeting bispecific COBRA was still able to lyse these cells in a T cell dependent cellular cytotoxicity assay. RT-PCR sequence analysis of these cells showed a major alternative transcript generated after CRISPR/Cas9, with Exon 1 and 3 spliced together in-frame, skipping Exon 2 completely, to express a truncated cell-surface receptor recognized by the EpCAM-COBRA. Researchers who use CRISPR/Cas9 must be cognizant of this potential to express alternative versions of their proteins and use sensitive orthogonal detection methods to ensure complete gene disruption.
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