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Functional characterizations of APP VAL604MET by CRISPR/CAS9
Author(s) -
Kim Danyeong,
Shim KyuHwan,
Van Vo Giau,
Bagyinszky Eva,
An Seong Soo
Publication year - 2020
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1002/alz.040123
Subject(s) - crispr , hek 293 cells , sanger sequencing , transfection , amyloid precursor protein , mutation , biology , cas9 , amyloid precursor protein secretase , amyloid beta , microbiology and biotechnology , gene , genetics , alzheimer's disease , disease , medicine , peptide , biochemistry , pathology
Background Many variants near the amyloid‐beta peptide (Aβ) of Amyloid Precursor Protein (APP) gene were reported major pathogenic mutations in Alzheimer's disease (AD), especially in early‐onset Alzheimer's disease (EOAD). Even though several mutations were investigated for the functional studies with cell and animal models, larger number of mutations were not investigated for the verification of the AD pathology. Previously, APP V604M was found from a EOAD patient with family history from Thailand. Since APP V604M was located outside of the Ab region, its causal pathogenic relationship could be questioned. In this study, APP V604M was introduced to a HEK293 cell line with CRISPR/Cas9 technology for the clarification of the functional pathogenicity. Method The guide RNA (gRNA) of APP V604M was introduced to a HEK293 cell line with CRISPR/Cas9 technology as previously reported. In CRISPR/Cas9, gRNA and donor DNA were designed for the APP V604M mutation and used to transfect HEK293 cells with Cas9 protein. The functional study of APP V604M was conducted using Aβ ELISA test. Aβ oligomers were measured by the Multimer Detection System (MDS). Result Transfection of APP V604M was confirmed using restriction enzymes. The mutation was identified by Sanger sequencing after single cell isolation. Next, the effects of the mutation were examined with gamma‐secretase activity, Aβ 42 productions and the presence of Aβ oligomers. MDS result showed the elevated levels of Aβ oligomers in APP V604M cells than the control, suggesting the mutation could be pathogenic. Additional biomarker results of gamma‐secretase activity and Aβ42 productions will be presented. Conclusion Transfection with CRISPR/Cas9 technology could help in understanding the pathogenicity of a mutation. APP V604M in HEK 293 was produced and biomarker study supported the pathogenicity.