PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington's disease.
PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington's disease.
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DOI：
10.1016/j.omtm.2022.08.005
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发表时间：
2022-09-08
期刊：
影响因子：
--
通讯作者：
Lee, Jong-Min
中科院分区：
文献类型：
--
作者：
Shin, Jun Wan;Hong, Eun Pyo;Park, Seri S.;Choi, Doo Eun;Zeng, Sophia;Chen, Richard Z.;Lee, Jong-Min
亨廷顿病(HD)是由亨廷顿蛋白(HTT)中CAG重复序列扩大引起的。由于HD占主导地位，而HTT的缺失会导致神经异常，因此安全的治疗策略需要选择性灭活突变的HTT。此前，我们提出了CRISPR-Cas9的概念，利用SNPs产生的突变特异性PAM位点选择性地灭活突变的HTT。为了揭示适合临床开发的靶点，我们分析了最大的HD基因数据集，以识别目标PAM改变SNPs(PAS)，并随后评估了它们的等位基因特异性。Rs2857935、rs16843804和rs16843836产生的基于PAM位点的gRNA在患者来源的细胞中显示出高水平的等位基因特异性。同时使用基于rs2857935-rs16843804或rs2857935-rs16843836的两个gRNA在突变型HTT中产生选择性基因组缺失，并阻止突变型HTT mRNA的转录，而不影响正常对应物的正常表达或切除片段在基因组其他地方的重新整合。RNA-SEQ和靶外分析证实了高水平的等位基因特异性和缺乏反复的靶外。大约60%的HD受试者符合突变特异性CRISPR-CAS9策略，该策略针对这三个PAS中的一个结合一个非等位基因特异性位点，支持基于PAS的等位基因特异性CRISPR方法在HD患者群体中的高度适用性。CRISPR-Cas9选择性灭活突变亨廷顿蛋白可能在亨廷顿病(HD)中产生强大的治疗效果。我们发现了在HD中允许等位基因特异性CRISPR-Cas9的PAM改变SNPs(rs2857935、rs16843804和rs16843836)。使用双gRNA组合靶向这些位点可以阻止突变的HTT mRNA的转录，而不会影响正常的对应物。
Huntington’s disease (HD) is caused by an expanded CAG repeat in huntingtin (HTT). Since HD is dominant and loss of HTT leads to neurological abnormalities, safe therapeutic strategies require selective inactivation of mutant HTT. Previously, we proposed a concept of CRISPR-Cas9 using mutant-specific PAM sites generated by SNPs to selectively inactivate mutant HTT. Aiming at revealing suitable targets for clinical development, we analyzed the largest HD genotype dataset to identify target PAM-altering SNPs (PAS) and subsequently evaluated their allele specificities. The gRNAs based on the PAM sites generated by rs2857935, rs16843804, and rs16843836 showed high levels of allele specificity in patient-derived cells. Simultaneous use of two gRNAs based on rs2857935-rs16843804 or rs2857935-rs16843836 produced selective genomic deletions in mutant HTT and prevented the transcription of mutant HTT mRNA without impacting the expression of normal counterpart or re-integration of the excised fragment elsewhere in the genome. RNA-seq and off-target analysis confirmed high levels of allele specificity and the lack of recurrent off-targeting. Approximately 60% of HD subjects are eligible for mutant-specific CRISPR-Cas9 strategies of targeting one of these three PAS in conjunction with one non-allele-specific site, supporting high applicability of PAS-based allele-specific CRISPR approaches in the HD patient population. CRISPR-Cas9 to selectively inactivate mutant huntingtin may produce robust therapeutic benefits in Huntington’s disease (HD). We revealed PAM-altering SNPs that permit allele-specific CRISPR-Cas9 in HD (rs2857935, rs16843804, and rs16843836). Targeting those sites in combinations using dual gRNAs prevented the transcription of mutant HTT mRNA without impacting the normal counterpart.
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