Francis P Pankowicz1, Mercedes Barzi1, Kang Ho Kim2, Xavier Legras1, Celeste Santos Martins1, Clavia Ruth Wooton-Kee2, William R Lagor3, Juan C Marini4, Sarah H Elsea5, Beatrice Bissig-Choisat1, David D Moore6, Karl-Dimiter Bissig7. 1. Center for Cell and Gene Therapy, Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, Texas; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas. 2. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas. 3. Center for Cell and Gene Therapy, Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, Texas; Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas. 4. Department of Pediatrics, Baylor College of Medicine, Houston, Texas. 5. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas. 6. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas; Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas. 7. Center for Cell and Gene Therapy, Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, Texas; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas; Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas. Electronic address: bissig@bcm.edu.
Abstract
BACKGROUND & AIMS: Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts. METHODS: In this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product. RESULTS: We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse. CONCLUSIONS: This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.
BACKGROUND & AIMS: Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts. METHODS: In this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product. RESULTS: We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse. CONCLUSIONS: This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.