Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype

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Abstract

We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.

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Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.

Gerald Schwank, Bon-Kyoung Koo, Valentina Sasselli … (2013)

Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect. Copyright © 2013 Elsevier Inc. All rights reserved.

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SNP Calling, Genotype Calling, and Sample Allele Frequency Estimation from New-Generation Sequencing Data

Rasmus Nielsen, Thorfinn Korneliussen, Anders Albrechtsen … (2012)

We present a statistical framework for estimation and application of sample allele frequency spectra from New-Generation Sequencing (NGS) data. In this method, we first estimate the allele frequency spectrum using maximum likelihood. In contrast to previous methods, the likelihood function is calculated using a dynamic programming algorithm and numerically optimized using analytical derivatives. We then use a Bayesian method for estimating the sample allele frequency in a single site, and show how the method can be used for genotype calling and SNP calling. We also show how the method can be extended to various other cases including cases with deviations from Hardy-Weinberg equilibrium. We evaluate the statistical properties of the methods using simulations and by application to a real data set.

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Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo.

Milton J. Finegold, Markus Grompe, Nicole Paulk … (2010)

Adeno-associated virus (AAV) vectors are ideal for performing gene repair due to their ability to target multiple different genomic loci, low immunogenicity, capability to achieve targeted and stable expression through integration, and low mutagenic and oncogenic potential. However, many handicaps to gene repair therapy remain. Most notable is the low frequency of correction in vivo. To date, this frequency is too low to be of therapeutic value for any disease. To address this, a point-mutation-based mouse model of the metabolic disease hereditary tyrosinemia type I was used to test whether targeted AAV integration by homologous recombination could achieve high-level stable gene repair in vivo. Both neonatal and adult mice were treated with AAV serotypes 2 and 8 carrying a wild-type genomic sequence for repairing the mutated Fah (fumarylacetoacetate hydrolase) gene. Hepatic gene repair was quantified by immunohistochemistry and supported with reverse transcription polymerase chain reaction and serology for functional correction parameters. Successful gene repair was observed with both serotypes but was more efficient with AAV8. Correction frequencies of up to 10(-3) were achieved and highly reproducible within typical dose ranges. In this model, repaired hepatocytes have a selective growth advantage and are thus able to proliferate to efficiently repopulate mutant livers and cure the underlying metabolic disease. AAV-mediated gene repair is feasible in vivo and can functionally correct an appropriate selection-based metabolic liver disease in both adults and neonates.