We have previously reported the genetic correction of Huntington’s disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR.
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Huntington’s disease (HD) is a dominantly inherited neurodegenerative disease caused by a CAG repeat expansion in the first exon of the gene Huntingtin (Htt). A dramatic pathological change in HD is the massive loss of striatal neurons as the disease progresses. A useful advance in HD would be the generation of a human-derived HD model to use for drug screening and understanding mechanisms of HD. We utilized the recently established human iPS cell line derived from HD patient fibroblasts to derive neuronal precursors and human striatal neurons. To achieve this goal, the differentiation of the HD-iPS cells into striatal fate required several steps. First, we generated nestin+/PAX6+/SOX1+/OCT4- neural stem cells (NSCs) from HD-iPS cells using the method of embryoid body formation. HD-NSCs were then subjected to a differentiation condition combining morphogens and neurotrophins to induce striatal lineage commitment. Striatal neuronal precursors/immature neurons stained with β-III tubulin, calbindin and GABA but not DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) were produced in this step. Finally, maturation and terminal differentiation of the striatal neuronal precursors/immature neurons resulted in striatal neurons expressing markers like DARPP-32. The HD-iPS cells derived striatal neurons and neuronal precursors contain the same CAG expansion as the mutation in the HD patient from whom the iPS cell line was established. Moreover, the HD-NSCs showed enhanced caspase activity upon growth factor deprivation compared to normal NSCs (from iPS or H9 NSCs). Therefore, these differentiated cells may produce a human HD cell model useful in the study of HD mechanisms and drug screening.