CRISPR based microRNA-122 strand specific knockout mice: Clinical characterization.

Maytal Gefen ¹ Alina Simerzin ¹ Mor Hindi ¹ Orr Levkovitch Siany ¹ Dayana Yaish ¹ Devora Gross ¹ Chofit Chai ¹ Nofar Rosenberg ¹ Zohar Shemuelian ¹ Boris Fehse ⁶ Ulrike Mock ⁶ Nicola Gagliani ⁵ Irm Hermans-Borgmeyer ⁴ Rebecca Haffner ² Shifra Ben-Dor ³ Eithan Galun ¹ Hilla Giladi ¹

¹Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Israel
²Veterinary Resources, Weizzman Institute, Israel
³Bioinformatics and Biological Computing Unit, Weizzman Institute, Israel
⁴Zentrum Für Molekulare Neurobiologie, University of Hamburg-Eppendorf (Uke), Germany
⁵Department of Medicine and Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Germany
⁶German Consortium for Translational Cancer Research (Dktk), German Cancer Research Center (Dkfz), Translational Gastrointestinal Oncology Group, Germany

MicroRNA-122, the dominant hepatocyte specific miRNA accounts for 70% of total liver miRNA population while undetectable in other tissues. miR-122 Knockout mice develop hepatic steatosis and fibrosis, culminating in HCC by 12 months of age. During microRNA biogenesis, one strand of the RNA duplex is selected for further processing and is termed the guide strand. The other strand, known as the microRNA* has recently been shown to be present in the cell. Interestingly most reports on miR-122 function do not address the function of the miR-122* strand. Recently, our lab has shown miR-122* to be significantly down regulated in human HCC tissues and to suppress tumorigenesis.

Our goal is to characterize the tumor suppressive function of each individual strand in a yet undocumented side by side study. We have generated five CRISPR/Cas9 transgenic mouse models. Using a point mutation approach, the seed of miR-122 or miR-122* was disrupted while maintaining the microRNA’s biogenesis and allowing for the complementary strand expression and function. Additionally, a model, where both seeds were mutated and two models with complete knock out of the microRNA region ranging approximately 60 bp each was generated as controls.

Differential gene expression between the seed specific knockout models, at different time points, will reveal the pathways involved in development of HCC, which we expect to determine by 12 months of age.