CRISPR/Cas9 editing of APP C-terminus attenuates \u03b2-cleavage and promotes \u03b1-cleavage

Jichao Sun,Lina Wang,Subhojit Roy,Krishanu Saha,Michael Stockton,Andrew J Petersen,Utpal Das,Anita Bhattacharyya,Mathew V Jones,Jonathan Loi,Andrew A Sproul,Jared Carlson-Stevermer,So Yeon Koo,Pamela J Mclean,Marion Delenclos,Xinyu Zhao,Dianhua Qiao,Minjie Shen,Amanda M Snead

doi:10.1038/s41467-018-07971-8

Abstract

CRISPR/Cas9 guided gene-editing is a potential therapeutic tool, however application to neurodegenerative disease models has been limited. Moreover, conventional mutation correction by gene-editing would only be relevant for the small fraction of neurodegenerative cases that are inherited. Here we introduce a CRISPR/Cas9-based strategy in cell and animal models to edit endogenous amyloid precursor protein (APP) at the extreme C-terminus and reciprocally manipulate the amyloid pathway, attenuating APP-β-cleavage and Aβ production, while up-regulating neuroprotective APP-α-cleavage. APP N-terminus and compensatory APP-homologues remain intact, with no apparent effects on neurophysiology in vitro. Robust APP-editing is seen in human iPSC-derived neurons and mouse brains with no detectable off-target effects. Our strategy likely works by limiting APP and BACE-1 approximation, and we also delineate mechanistic events that abrogates APP/BACE-1 convergence in this setting. Our work offers conceptual proof for a selective APP silencing strategy.

Highlights

CRISPR/Cas[9] guided gene-editing is a potential therapeutic tool, application to neurodegenerative disease models has been limited
As a control for assay-validation, we found that a C-terminus deletion abrogated amyloid precursor protein (APP)/ BACE-1 complementation[10]; in line with previous studies showing that deletions/mutations of the APP C-terminus can attenuate Aβ production[11,12,13]
To compare the editing efficiency of these single guide-RNA (sgRNA), we engineered a stable H4 neuroglioma cell line expressing single copies of APP:VN and BACE-1:VC (APP/ BACEsingle_copy), where editing efficiency of a given sgRNA could be determined as a simple on/off fluorescence readout and the effect of APP truncation could be assessed by evaluating secreted Aβ

Summary

Introduction

CRISPR/Cas[9] guided gene-editing is a potential therapeutic tool, application to neurodegenerative disease models has been limited. We introduce a CRISPR/Cas9-based strategy in cell and animal models to edit endogenous amyloid precursor protein (APP) at the extreme C-terminus and reciprocally manipulate the amyloid pathway, attenuating APP-β-cleavage and Aβ production, while up-regulating neuroprotective APP-α-cleavage. Strategies aimed at correcting point-mutations would only be applicable to the small fraction of neurodegenerative diseases that are inherited (typically < 10% of cases). Our broad idea is to rationally edit small segments of endogenous proteins known to play key roles in the progression of disease, with the ultimate goal of attenuating their pathologic activity. Using CRISPR-tools, cell/molecular biology, live imaging, deep sequencing, electrophysiology and in vivo animal studies, here we highlight a strategy to favorably manipulate the amyloid pathway by gene editing

Methods

Results

Conclusion