<br>CRISPR-Cas9 介导的病毒 E1A 基因靶向抑制腺病毒复制,Molecular Therapy - Nucleic Acids

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CRISPR-Cas9 介导的病毒 E1A 基因靶向抑制腺病毒复制
Molecular Therapy - Nucleic Acids ( IF 6.5 ) Pub Date : 2023-03-03 , DOI: 10.1016/j.omtn.2023.02.033
Zrinka Didara ₁ , Florian Reithofer ₁ , Karina Zöttl ₁ , Alexander Jürets ₁ , Izabella Kiss ₁ , Angela Witte ₂ , Reinhard Klein ₁

Affiliation

靶向 DNA 的 CRISPR-Cas 系统能够切割哺乳动物细胞中的 dsDNA。因此，它们已被用于靶向 dsDNA 病毒的基因组，主要是当它们存在于非复制状态且拷贝数低的细胞中时。然而，某些裂解复制病毒在很短的时间内产生的大量病毒 DNA 可能会使 CRISPR-Cas 系统的能力达到极限。病毒 DNA 复制位点的可及性、封装前 DNA 的可及性短或其与屏蔽蛋白的复合是进一步的潜在障碍。腺病毒是快速复制的 dsDNA 病毒，目前尚无批准的抗病毒疗法。我们通过用一组向导 RNA 靶向其主调节因子 E1A 来评估 CRISPR-Cas9 在体外抑制人腺病毒 5 复制的效力，并观察到感染性病毒颗粒减少了多达三个数量级。靶 DNA 切割也对感染周期中积累的病毒 DNA 量产生负面影响。这一结局主要是由靶位点之间发生的特异性缺失、倒位和重复引起的，在大多数情况下，这消除了大多数 E1A 功能。此外，我们比较了两种多重 gRNA 表达策略并获得了可比的结果。

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Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene

DNA-targeting CRISPR-Cas systems are able to cleave dsDNA in mammalian cells. Accordingly, they have been employed to target the genomes of dsDNA viruses, mostly when present in cells in a non-replicative state with low copy numbers. However, the sheer amount of viral DNA produced within a very short time by certain lytically replicating viruses potentially brings the capacities of CRISPR-Cas systems to their limits. The accessibility of viral DNA replication sites, short time of accessibility of the DNA before encapsidation, or its complexation with shielding proteins are further potential hurdles. Adenoviruses are fast-replicating dsDNA viruses for which no approved antiviral therapy currently exists. We evaluated the potency of CRISPR-Cas9 in inhibiting the replication of human adenovirus 5 in vitro by targeting its master regulator E1A with a set of guide RNAs and observed a decrease in infectious virus particles by up to three orders of magnitude. Target DNA cleavage also negatively impacted the amount of viral DNA accumulated during the infection cycle. This outcome was mainly caused by specific deletions, inversions, and duplications occurring between target sites, which abolished most E1A functions in most cases. Additionally, we compared two strategies for multiplex gRNA expression and obtained comparable results.

更新日期：2023-03-03

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