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Abstract: TH-PO796

CRISPR/Cas9 Zebrafish Models Do Not Recapitulate 4 Human Monogenic Causes of Nephrotic Syndrome

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Deutsch, Konstantin, Boston Children's Hospital, Boston, Massachusetts, United States
  • Schneider, Ronen, Boston Children's Hospital, Boston, Massachusetts, United States
  • Kitzler, Thomas Michael, Boston Children's Hospital, Boston, Massachusetts, United States
  • Jobst-Schwan, Tilman, Boston Children's Hospital, Boston, Massachusetts, United States
  • Kolvenbach, Caroline M., Boston Children's Hospital, Boston, Massachusetts, United States
  • Kause, Franziska, Boston Children's Hospital, Boston, Massachusetts, United States
  • Hildebrandt, Friedhelm, Boston Children's Hospital, Boston, Massachusetts, United States
Background

Steroid-resistant nephrotic syndrome (SRNS) is characterized by proteinuria due to disruption of the glomerular filtration barrier and is a frequent cause of chronic kidney disease. Currently, more than 60 monogenic causes of human SRNS are known. Historically, zebrafish have been a commonly used animal model for human monogenic SRNS, using morpholino oligonucleotides. Recently, generation of zebrafish shifted to CRISPR-mediated knockout (KO), which often showed lack of recapitulation of the human disease phenotype in zebrafish (Dev Cell 32:97, 2015). We recapitulated the human SRNS phenotype due to Magi2 mutation (Nat Commun 9:1980, 2018) in a CRISPR model of magi2a (Kidney Int 95:1079, 2019). Recently, we discovered recessive mutations as novel causes of human SRNS in the following genes: advillin (AVIL) (J Clin Invest 127:4257, 2017), PR/SET domain 15 (PRDM15), deleted in liver cancer 1 (DLC1) and Intersectin1 (ITSN1) (Nat Commun 9:1980, 2018).

Methods

To recapitulate monogenic causes of human SRNS and to study developmental phenotypes by CRISPR/Cas9 KO of the zebrafish orthologues avil, dlc1, itsn1 and prdm15, we performed microinjections of multiple guide RNAs for each of the genes and generated stable KO zebrafish lines for these genes. Survival curves as well as phenotye assessments were generated for acute knockdown (KD) and stable KO zebrafish by monitoring larvae for 21 days.

Results

To assess larval-onset phenotype, we generated acute KD animals, which did not show a significant difference in survival or phenotype compared to animals injected with a non-binding control guide RNA and wildtype animals. We subsequently generated stable KO lines with 4 different KO-alleles for AVIL, 3 for DLC1, 4 for ITSN1 and 6 for PRDM15 respectively. None of these alleles showed a significant phenotypical difference in homozygous fish compared to heterozygous and wildtype controls.

Conclusion

Failure to recapitulate disease phenotypes in CRISPR models is currently predominantly attributed to upregulation of paralogues, triggered by mRNA degradation (Nature 568:197, 2019). In line with these results, CRISPR/Cas9 zebrafish models of the human SRNS genes AVIL, DLC1, PRDM15 and ITSN1 do not recapitulate the human phenotype of nephrotic syndrome and do not impede zebrafish larval survival.

Funding

  • NIDDK Support