Kidney Week

Abstract: SA-PO577

A Microhomology-Mediated End-Joining (MMEJ)-Based F0 Assay in Zebrafish for Rapid Screening of Genetic Modifiers of Kidney Cysts

Session Information

• Cystic Kidney Diseases: Genetic Causes, Modifiers, and Extrarenal Manifestations
  October 26, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic

Authors

• Zhu, Ping, Mayo Foundation for Medical Education and Research, Rochester, Minnesota, United States

Ping Zhu

• Xu, Xiaolei, Mayo Foundation for Medical Education and Research, Rochester, Minnesota, United States

Xiaolei Xu, PhD

• Lin, Xueying, Mayo Foundation for Medical Education and Research, Rochester, Minnesota, United States

Xueying Lin, PhD

Background

Autosomal-dominant polycystic kidney disease (ADPKD) is one of the most prevalent and potentially lethal genetic diseases. ADPKD patients exhibit highly variable phenotypes, of which genetic modifiers are thought to play an important role. However, the identity of the genetic modifiers remains largely unknown because of the lack of an efficient method. Prompted by the success of applying microhomology-mediated end joining (MMEJ)-based genome editing technology for the identification of genetic modifiers of cardiomyopathy in the F0 generation, we decided to explore the F0-based genetic assay for discovering genetic modifiers of ADPKD.

Methods

We generated zebrafish mutants of IFT140, a newly identified ADPKD gene. All ift140 mutants exhibited distinctive pronephric cysts and were otherwise normal till approximately 2 weeks of age. Injection of a MMEJ-inducing sgRNA against ift140 consistently resulted in pronephric cysts in >90% of the injected embryos, and the cysts can be easily scored under a light microscope at 4-6 dpf. To test the feasibility of developoing a modifier screen platform, we co-injected MMEJ-inducing sgRNAs targeting ift140 and a gene of interest into one-cell staged wild type embryos and assessed kidney cysts at 5 dpf.

Results

In a pilot screen, we tested 18 genes that are implicated in the signaling pathways known to be dysregulated in ADPKD. Inactivation of genes that were previously suggested beneficial, such as mtor, was found to reduce the number of ift140^MJ embryos with pronephric cysts. By contrast, inactivation of genes that were previously suggested detrimental did not suppress pronephric cyst formation. Besides genes with known modifying effects, novel protective modifiers such as ulk1a have been suggested from our pilot screen. Using mtor and ulk1a stable mutants, we confirmed the protective modifying effects of mTor and Ulk1a inhibition in ift140-based as well as pkd1-based kidney cyst development.

Conclusion

Thus, ift140^MJ embryo can be used as a surrogate to rapidly discover candidate protective modifiers of kidney cysts, which can be validated by generating stable mutants and extended to pkd1-based cystogenesis.

Funding

• NIDDK Support