Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System

Abstract

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.

Genome Editing of Structural Variations: Modeling and Gene Correction

“…The HD phenotype was reversed when brain-derived neurotrophic factor (BDNF) expression and caspase activity were evaluated from the corrected iPSC-derived neuronal cells, and the differentiated cells showed survival when they were grafted into the striatum of a HD mouse model. However, a following experiment using Cas9, Cas9-nickase, or TALEN showed high efficiency of insertion of CAG repeats into the HTT gene exon 1 in human cells [92].…”

“…The use of engineered nucleases enables the generation of disease models by conveniently introducing DNA repeats into a specific region [92], removing disease-causing expanded DNA repeats [93,94], or neutralizing the effect of expanded DNA repeats [95].…”

Using Genome Engineering to Understand Huntington’s Disease

“…For polyglutamine disease, it is also possible to detect the prevalence of the polyglutamine expansion through the use of specific antibodies, which detect the expanded polyglutamine region (Fig. 4; An et al 2014). The ability to qualitatively assess how many cells have been corrected will increase the field's understanding of what may be a therapeutic level of correction for the disease.…”

Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas [Review]

“…Because mutation of the DNMT3B gene causes immunodeficiency, centromeric region instability, and facial anomalies (ICF) syndrome in humans, these mutant iPSC lines are a good disease model. Various disease models, such as a Huntington's disease model [20] and a HIV resistance model [21], have been generated from human iPSCs using CRISPR/ Cas systems. In addition, gene corrections mediated by HR using CRISPR/Cas have been reported in models of diseases such as β-thalassemia, muscular dystrophy, and sickle cell disease [22][23][24][25][26].…”