Allele-specific silencing of the gain-of-function mutation in Huntington's disease using CRISPR/Cas9.
Allele-specific silencing of the gain-of-function mutation in Huntington's disease using CRISPR/Cas9.
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DOI：
10.1172/jci.insight.141042
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发表时间：
2022-10-10
期刊：
影响因子：
8
通讯作者：
Lee, Jong-Min
中科院分区：
文献类型：
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作者：
Shin, Jun Wan;Hong, Eun Pyo;Choi, Doo Eun;Seong, Ihn Sik;Park, Seri S.;Whittaker, Madelynn N.;Kleinstiver, Benjamin P.;Chen, Richard Z.;Lee, Jong-Min
亨廷顿病（HD）中的显性功能获得机制表明，选择性沉默突变HTT产生强大的治疗益处。在这里，利用外显子前间区序列邻近基序改变（PAM改变）SNP（PAS），我们开发了等位基因特异性CRISPR/Cas9策略，以通过无义介导的衰变（NMD）永久地抑制突变HTT。综合序列/单倍型分析鉴定了HD受试者中常见HTT单倍型外显子上的SNP生成的NGG PAM位点，揭示了临床相关的基于PAS的突变体特异性CRISPR/Cas9策略。rs363099的替代等位基因（第29外显子）消除了HD中最常见的正常HTT单倍型上的NGG PAM位点，允许在预测的约20%的具有欧洲血统的HD受试者中进行突变体特异性CRISPR/Cas9治疗。我们的基于rs363099的CRISPR/Cas9在患者来源的细胞中显示出完美的等位基因特异性和良好的靶向效率。突变体HTT mRNA的显著减少和突变体蛋白的完全丧失表明我们的等位基因特异性CRISPR/Cas9策略通过NMD使突变体HTT失活。此外，GUIDE-Seq分析和随后的验证实验支持高水平的靶基因特异性。我们的数据证明了显著的靶向群体，完全的突变特异性，在患者来源的细胞中的良好靶向效率，以及对蛋白质编码基因的最小脱靶效应，证明了基于PAS的等位基因特异性NMD-CRISPR/Cas9的概念，并支持其在HD中的治疗潜力。
Dominant gain-of-function mechanisms in Huntington’s disease (HD) suggest that selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic protospacer adjacent motif–altering (PAM-altering) SNP (PAS), we developed an allele-specific CRISPR/Cas9 strategy to permanently inactivate mutant HTT through nonsense-mediated decay (NMD). Comprehensive sequence/haplotype analysis identified SNP-generated NGG PAM sites on exons of common HTT haplotypes in HD subjects, revealing a clinically relevant PAS-based mutant-specific CRISPR/Cas9 strategy. Alternative allele of rs363099 (29th exon) eliminates the NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant-specific CRISPR/Cas9 therapeutics in a predicted ~20% of HD subjects with European ancestry. Our rs363099-based CRISPR/Cas9 showed perfect allele specificity and good targeting efficiencies in patient-derived cells. Dramatically reduced mutant HTT mRNA and complete loss of mutant protein suggest that our allele-specific CRISPR/Cas9 strategy inactivates mutant HTT through NMD. In addition, GUIDE-Seq analysis and subsequent validation experiments support high levels of on-target gene specificity. Our data demonstrate a significant target population, complete mutant specificity, decent targeting efficiency in patient-derived cells, and minimal off-target effects on protein-coding genes, proving the concept of PAS-based allele-specific NMD-CRISPR/Cas9 and supporting its therapeutic potential in HD.
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影响因子：
46.9
作者：
Jackson, AL;Bartz, SR;Linsley, PS
通讯作者：
Linsley, PS
影响因子：
4.5
作者：
Dietrich P;Johnson IM;Alli S;Dragatsis I
通讯作者：
Dragatsis I
DOI：
10.1073/pnas.1706193114
发表时间：
2017-10-17
影响因子：
11.1
作者：
Jain, Ankur;Zode, Gulab;Sheffield, Val C.
通讯作者：
Sheffield, Val C.
影响因子：
16.2
作者：
Incontro, Salvatore;Asensio, Cedric S.;Edwards, Robert H.;Nicoll, Roger A.
通讯作者：
Nicoll, Roger A.
影响因子：
23.9
作者：
通讯作者：
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