CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs

Lixia Wang,Keiichiro Suzuki,Lina Fu,Liang Sun,Si Wang,Zhaoxia Wang,Yang Yu,Xiuling Xu,Fei Yi,Guang-Hui Liu,Jie Qiao,Jiping Yang,Ze Yang,Juan Carlos Izpisua Belmonte,Jing Qu,Yun Yuan

doi:10.1007/s13238-017-0397-3

Abstract

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease with cellular and molecular mechanisms yet to be fully described. Mutations in a number of genes including SOD1 and FUS are associated with familial ALS. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts of familial ALS patients bearing SOD1+/A272C and FUS+/G1566A mutations, respectively. We further generated gene corrected ALS iPSCs using CRISPR/Cas9 system. Genome-wide RNA sequencing (RNA-seq) analysis of motor neurons derived from SOD1+/A272C and corrected iPSCs revealed 899 aberrant transcripts. Our work may shed light on discovery of early biomarkers and pathways dysregulated in ALS, as well as provide a basis for novel therapeutic strategies to treat ALS.

Highlights

Amyotrophic lateral sclerosis (ALS) are a group of progressive but fatal neurodegenerative diseases due to the selective loss of functional motor neurons in the brain and spinal cord
The generated induced pluripotent stem cell (iPSC) displayed typical pluripotent stem cell-like morphology, expressed pluripotency markers, such as OCT4, NANOG and SOX2, formed teratomas consisting of three germ-layers in vivo, maintained unmethylated CpG islands in the promoter of OCT4, and demonstrated normal karyotypes (Fig. 1B–F)
DNA sequencing results demonstrated the presence of superoxide dismutase 1 (SOD1)+/A272C or FUS+/G1566A mutations in the two lines of ALS iPSCs, respectively (Fig. 1G)

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) are a group of progressive but fatal neurodegenerative diseases due to the selective loss of functional motor neurons in the brain and spinal cord. Excitotoxic/Inflammatory/Oxidative insults, misfolded proteins and aggregates, aberrant RNA processing, unstable genome, and mitochondria dysfunction have all been implicated in ALS (Pasinelli and Brown, 2006; Kiskinis et al, 2014). Many genes have been implicated in FALS, such as SOD1, FUS, TDP43, C9ORF72, VAPB, etc. Mutant SOD1 was found as aberrant misfolded aggregates in FALS (Bruijn et al, 1998; Bosco et al, 2010). FUS was initially identified in cancer and encodes a RNA/DNA binding protein (Crozat et al, 1993; Baechtold et al, 1999). A number of mutations in FUS gene were identified in ALS cases, like A1564G, C1574T, G1566A, etc. Aggregates containing mutant FUS protein have been found in motor neurons from ALS patients by postmortem analysis (Vance et al, 2009)

Methods

Results

Conclusion