通过 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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通过 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

DNA 靶向 CRISPR-Cas 系统能够切割哺乳动物细胞中的 dsDNA。因此，它们已被用来靶向双链DNA病毒的基因组，主要是当双链DNA病毒以低拷贝数存在于非复制状态的细胞中时。然而，某些裂解性复制病毒在很短的时间内产生的大量病毒 DNA 可能使 CRISPR-Cas 系统的能力达到极限。病毒DNA复制位点的可及性、衣壳化前DNA的可及性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。我们评估了 CRISPR-Cas9 通过用一组引导 RNA 靶向其主调控因子来抑制人类腺病毒 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。目标 DNA 切割也会对感染周期中积累的病毒 DNA 量产生负面影响。这一结果主要是由目标位点之间发生的特定删除、倒位和重复引起的，这在大多数情况下废除了大多数 E1A 功能。此外，我们还比较了两种多重 gRNA 表达策略并获得了可比较的结果。

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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 by targeting its master regulator 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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