Keratinocytes sense and eliminate CRISPR DNA through STING/IFN-κ activation and APOBEC3G induction.
Keratinocytes sense and eliminate CRISPR DNA through STING/IFN-κ activation and APOBEC3G induction.
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DOI：
10.1172/jci159393
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发表时间：
2023-05-01
影响因子：
15.9
通讯作者：
Gudjonsson, Johann E.
中科院分区：
文献类型：
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作者：
Sarkar, Mrinal K.;Uppala, Ranjitha;Zeng, Chang;Billi, Allison C.;Tsoi, Lam C.;Kidder, Austin;Xing, Xianying;White, Bethany E. Perez;Shao, Shuai;Plazyo, Olesya;Sirobhushanam, Sirisha;Xing, Enze;Jiang, Yanyun;Gallagher, Katherine A.;Voorhees, John J.;Kahlenberg, J. Michelle;Gudjonsson, Johann E.
CRISPR/Cas9已被提议作为遗传性皮肤病的治疗方法。在这里，我们报告了CRISPR转染激活角质形成细胞中的STING依赖性抗病毒反应，通过诱导IFN-κ导致内源性干扰素（IFN）反应增强，导致继发于胞苷脱氨酶基因APOBEC 3G诱导的质粒稳定性降低。值得注意的是，CRISPR产生的KO角质形成细胞对IFN-κ和IFN刺激基因（ISG）表达具有永久性抑制，继发于DNA甲基转移酶DNMT 3B对IFNK启动子区域的超甲基化。在CRISPR转染之前通过baricitinib进行JAK抑制增加了转染效率，防止了IFNK启动子高甲基化，并恢复了正常的IFN-κ活性和ISG应答。这项工作表明，CRISPR介导的基因校正改变了角质形成细胞中的抗病毒反应，对未来使用CRISPR技术治疗遗传性皮肤病的基因疗法具有影响，并表明药理学JAK抑制作为促进和减弱CRISPR/Cas9治疗方法中无意选择效应的工具。
CRISPR/Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ, leading to decreased plasmid stability secondary to induction of the cytidine deaminase gene APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies for inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR/Cas9 therapeutic approaches.