Efficient introduction of disease-related mutations into human iPSCs using CRISPR/Cas9 to model Alzheimer’s disease
CRISPR/Cas9 allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, e.g. in human pluripotent stem cells. Although CRISPR/Cas9 is used extensively to engineer gene knock-outs, precise editing using CRISPR/Cas9 remains inefficient, preventing its widespread use for modeling genetic disorders by introducing disease-associated mutations. We developed a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed “CORRECT” for scarless editing. Using this approach, we generated human induced pluripotent stem cells (iPSCs) with heterozygous and homozygous dominant early onset Alzheimer’s disease (EOAD) mutations in amyloid precursor protein (APPSwe) and presenilin 1 (PSEN1M146V) and derive cortical neurons, which display genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of disease-associated mutations with CRISPR/Cas9, facilitating the study of human dementia.
Date: 23 March 2017, 12:00 (Thursday, 10th week, Hilary 2017)
Venue: Department of Psychiatry, Headington OX3 7JX
Venue Details: Seminar Room
Speaker: Dylan Kwart (The Rockefeller University)
Organising department: Department of Psychiatry
Organiser: Corinne Prescott (University of Oxford)
Organiser contact email address: corinne.prescott@psych.ox.ac.uk
Host: Professor Simon Lovestone, (Dept of Psychiatry, University of Oxford)
Booking required?: Not required
Audience: Members of the University only
Editor: Corinne Prescott