Precise and efficient insertion of A673T mutation in APP gene using MSYM

Abstract

Abstract Alzheimer's Disease is a progressive neurodegenerative disorder characterized by toxic accumulation of amyloid beta peptides and tau neurofibrillary tangles. A673T mutation, as a protective mutation existing in natural people has a huge potential in the pharmacotherapy of Alzheimer's Disease, particularly for the combination with stem cell therapy, which may not only provide clinic benefit to amyloid beta peptides, but correct irreversible injury due to amyloid beta peptides. Here we describe a CRISPR/Cas9-based genome-editing framework that allows point mutation with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by increasing same sense mutation positioned in the guide RNA target sequence along with cleavage site, and establish a method termed “MSYM” for scarless genome editing. Using this approach, we generated the A673T mutation in iPS cells. This point mutation is an alanine-to-threonine substitution at position 673 of APP. More importantly, this approach provides more options for researchers and clinicians in efficient introduction of specific sequence changes, facilitating study of human disease, not only AD.