CRISPR Activation of PKD1 in Immortalized Cell Lines Is Limited by Its Heterochromatinized Proximal Promoter
- Genetic Diseases: Cystic - Basic
November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1201 Genetic Diseases of the Kidneys: Cystic
- Chakraborty, Anubhav, University of Kansas Medical Center, Kansas City, Kansas, United States
- Ward, Christopher J., University of Kansas Medical Center, Kansas City, Kansas, United States
- Yu, Alan S.L., University of Kansas Medical Center, Kansas City, Kansas, United States
Loss of function mutations in PKD1 are the predominant causes of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is characterized by the progressive enlargement of renal cysts which leads to a decline in renal function. Cysts form when the functional levels of polycystin 1 (PC1), the protein product of PKD1, decrease below a critical threshold. We hypothesize that increasing the expression of PKD1 could potentially restore functional levels of PC1 and rescue the disease.
To increase the expression of PKD1 using CRISPR activation (CRISPRa), we designed and screened several guide RNAs (gRNA) targeting the proximal promoter of the gene in mouse renal cortical collecting duct (M1) and HEK293T cell lines. We measured the abundance of PKD1 transcripts in these cell lines using qRTPCR and assessed the chromatin accessibility of the PKD1 proximal promoter.
CRISPRa-mediated PKD1 upregulation in M1 or HEK293T cells reached a maximum of 2-2.5-fold (p<0.001) using pooled gRNAs targeting the 100 bp region upstream of the transcriptional start site. In contrast, positive control genes (Klf1, Nkx2, Oct4, INS, and TTN1) displayed substantially greater increases in expression (5-6000-fold). Both cell lines exhibited low PKD1 mRNA abundance (~10 copies/cell). PCR-based chromatin accessibility assay showed less than a 4-fold enrichment in the PKD1 proximal promoter (in both cell lines), indicating a heterochromatinized region. Additionally, H3K27 acetylation and DNase hypersensitivity data from ENCODE showed that heterochromatization of the mouse or human PKD1 proximal promoter is common to most immortalized cell lines.
Our findings suggest that the heterochromatinized PKD1 proximal promoter poses a limitation on the extent to which the gene can be effectively upregulated by CRISPRa. Understanding these constraints is crucial for developing strategies to overcome heterochromatinization and enhance PKD1 expression, potentially enabling therapeutic interventions for ADPKD.