TY - JOUR
T1 - Targeting N-Terminal Huntingtin with a Dual-sgRNA Strategy by CRISPR/Cas9
AU - Wu, Junjiao
AU - Tang, Yu
AU - Zhang, Chun Li
N1 - Publisher Copyright:
© 2019 Junjiao Wu et al.
PY - 2019
Y1 - 2019
N2 - Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder, caused by a CAG/polyglutamine (polyQ) repeat expansion in the Huntingtin (HTT) gene. The polyQ tract is located in and transcribed from N-terminal HTT of exon 1. HTT is a large multifaceted protein, which participates in a range of cellular functions. Previous studies have shown that truncated HTT, which lacks N-terminus, retains specific functions that can produce neuroprotective benefits. It gives an insight that it is possible to repair HD by removing deleterious N-terminal HTT with CRISPR/Cas9, without compromising functions of remaining HTT peptides. To successfully generate functional truncated HTT proteins, an alternative downstream ATG start codon that is capable of initiating truncated HTT expression is required. In this study, we searched all possible in-frame ATGs before exon 7 and demonstrated that one of them can indeed initiate the downstream GFP expression in plasmids. We then tried to remove endogenous N-terminal HTT with an optimized dual-sgRNA strategy by CRISPR/Cas9; however, we cannot detect obvious traits of truncated HTT expression. Our results suggest that noncanonical ATGs of N-terminal HTT may not be effective in the genomic context, as in the construct context. Nevertheless, our study examined the therapeutic efficacy of downstream noncanonical ATGs for protein translation and also provided an optimized dual-sgRNA strategy for further genome manipulation of the HTT gene.
AB - Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder, caused by a CAG/polyglutamine (polyQ) repeat expansion in the Huntingtin (HTT) gene. The polyQ tract is located in and transcribed from N-terminal HTT of exon 1. HTT is a large multifaceted protein, which participates in a range of cellular functions. Previous studies have shown that truncated HTT, which lacks N-terminus, retains specific functions that can produce neuroprotective benefits. It gives an insight that it is possible to repair HD by removing deleterious N-terminal HTT with CRISPR/Cas9, without compromising functions of remaining HTT peptides. To successfully generate functional truncated HTT proteins, an alternative downstream ATG start codon that is capable of initiating truncated HTT expression is required. In this study, we searched all possible in-frame ATGs before exon 7 and demonstrated that one of them can indeed initiate the downstream GFP expression in plasmids. We then tried to remove endogenous N-terminal HTT with an optimized dual-sgRNA strategy by CRISPR/Cas9; however, we cannot detect obvious traits of truncated HTT expression. Our results suggest that noncanonical ATGs of N-terminal HTT may not be effective in the genomic context, as in the construct context. Nevertheless, our study examined the therapeutic efficacy of downstream noncanonical ATGs for protein translation and also provided an optimized dual-sgRNA strategy for further genome manipulation of the HTT gene.
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U2 - 10.1155/2019/1039623
DO - 10.1155/2019/1039623
M3 - Article
C2 - 31828084
AN - SCOPUS:85075714091
SN - 2314-6133
VL - 2019
JO - BioMed Research International
JF - BioMed Research International
M1 - 1039623
ER -