利用 TAR 特异性 CRISPR 工具沉默整合的 SIV 前病毒 DNA。
Silencing integrated SIV proviral DNA with TAR-specific CRISPR tools.
机构信息
Host-Pathogen Interactions Program, Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, Texas, USA.
Department of Microbiology, Immunology, and Molecular Genetics, UT Health San Antonio, San Antonio, Texas, USA.
出版信息
J Med Primatol. 2020 Oct;49(5):269-279. doi: 10.1111/jmp.12494. Epub 2020 Sep 9.
BACKGROUND
One approach for a functional HIV cure is to prevent transcription from integrated proviral DNA. A critical step in HIV transcription is the Tat protein interaction with the TAR element viral RNA. We tested the strategy of blocking this Tat-TAR interaction in the SIVmac model.
METHODS
We designed five CRISPR short guiding RNAs (sgRNAs) targeting the SIVmac TAR element, along with inactive versions of Cas9 (dCas9). These sgRNA constructs were delivered as ribonucleoproteins or plasmid DNA, along with SIV DNA. The constructs were also tested in integrated viral DNA in a cell line chronically infected by SIV.
RESULTS
The sgRNAs targeting the coding strand of the TAR element inhibited SIV RNA transcription in association with dCas9-KRAB, but not with dCas9.
CONCLUSIONS
Induction of epigenetic modifications may be more effective in inactivating provirus than transcriptional interference and thus may be a better strategy to achieve a functional cure in vivo.
背景
功能性 HIV 治愈方法之一是防止整合的前病毒 DNA 转录。HIV 转录的关键步骤是 Tat 蛋白与 TAR 元件病毒 RNA 的相互作用。我们在 SIVmac 模型中测试了阻断这种 Tat-TAR 相互作用的策略。
方法
我们设计了五个针对 SIVmac TAR 元件的 CRISPR 短引导 RNA(sgRNA)，以及无活性的 Cas9(dCas9)。这些 sgRNA 构建体与 SIV DNA 一起作为核糖核蛋白或质粒 DNA 递送。这些构建体也在受 SIV 慢性感染的细胞系中的整合病毒 DNA 中进行了测试。
结果
靶向 TAR 元件编码链的 sgRNA 与 dCas9-KRAB 一起抑制了 SIV RNA 的转录，但与 dCas9 一起则没有。
结论
诱导表观遗传修饰可能比转录干扰更有效地使前病毒失活，因此可能是在体内实现功能性治愈的更好策略。
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