Transient modulation of the genes involved in immunity, without exerting a permanent change in the DNA code, can be an effective strategy to modulate the course of many inflammatory conditions. CRISPR-Cas9 technology represents a promising platform for achieving this goal. Truncation of guide RNA (gRNA) from the 5′ end enables the application of a nuclease competent Cas9 protein for transcriptional modulation of genes, allowing multifunctionality of CRISPR. Here, we introduce an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo. In this repressor system, two transcriptional repressors—heterochromatin protein 1 (HP1a) and Krüppel-associated box (KRAB)—are fused to the MS2 coat protein and subsequently recruited by gRNA aptamer binding to a nuclease competent CRISPR complex containing truncated gRNAs. With the enhanced repressor, we demonstrate transcriptional repression of the Myeloid differentiation primary response 88 (Myd88) gene in vitro and in vivo. We demonstrate that this strategy can efficiently downregulate Myd88 expression in lung, blood and bone marrow of Cas9 transgenic mice that receive systemic injection of adeno-associated virus (AAV)2/1-carrying truncated gRNAs targeting Myd88 and the MS2-HP1a-KRAB cassette. This downregulation is accompanied by changes in downstream signalling elements such as TNF-α and ICAM-1. Myd88 repression leads to a decrease in immunoglobulin G (IgG) production against AAV2/1 and AAV2/9 and this strategy modulates the IgG response against AAV cargos. It improves the efficiency of a subsequent AAV9/CRISPR treatment for repression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a gene that, when repressed, can lower blood cholesterol levels. We also demonstrate that CRISPR-mediated Myd88 repression can act as a prophylactic measure against septicaemia in both Cas9 transgenic and C57BL/6J mice. When delivered by nanoparticles, this repressor can serve as a therapeutic modality to influence the course of septicaemia. Collectively, we report that CRISPR-mediated repression of endogenous Myd88 can effectively modulate the host immune response against AAV-mediated gene therapy and influence the course of septicaemia. The ability to control Myd88 transcript levels using a CRISPR-based synthetic repressor can be an effective strategy for AAV-based CRISPR therapies, as this pathway serves as a key node in the induction of humoral immunity against AAV serotypes.

对参与免疫的基因进行瞬时调节，而不对 DNA 密码产生永久性改变，可能是调节许多炎症性疾病进程的有效策略。 CRISPR-Cas9 技术代表了实现这一目标的一个有前景的平台。从 5' 端截断向导 RNA (gRNA) 使得能够应用具有核酸酶活性的 Cas9 蛋白进行基因转录调节，从而实现 CRISPR 的多功能性。在这里，我们引入了一种增强的基于 CRISPR 的转录阻遏蛋白，可以在体内重新编程免疫稳态。在该阻遏蛋白系统中，两个转录阻遏蛋白——异染色质蛋白 1 (HP1a) 和 Krüppel 相关盒 (KRAB)——与 MS2 外壳蛋白融合，随后通过 gRNA 适体与含有截短 gRNA 的核酸酶活性 CRISPR 复合物结合来招募。通过增强的阻遏蛋白，我们在体外和体内证明了骨髓分化初级反应 88 ( Myd88 ) 基因的转录抑制。我们证明，该策略可以有效下调 Cas9 转基因小鼠的肺、血液和骨髓中Myd88 的表达，这些小鼠接受全身注射腺相关病毒 (AAV)2/1 携带靶向Myd88和 MS2-HP1a-KRAB 盒的截短 gRNA 。这种下调伴随着下游信号元件（例如TNF-α和ICAM-1）的变化。 Myd88抑制导致针对 AAV2/1 和 AAV2/9 的免疫球蛋白 G (IgG) 产生减少，并且该策略调节针对 AAV 货物的 IgG 反应。 它提高了后续 AAV9/CRISPR 治疗抑制前蛋白转化酶枯草杆菌蛋白酶/kexin 9 型 ( PCSK9 ) 的效率，该基因在受到抑制时可以降低血液胆固醇水平。我们还证明，CRISPR 介导的Myd88抑制可以作为 Cas9 转基因小鼠和 C57BL/6J 小鼠败血症的预防措施。当通过纳米颗粒传递时，这种阻遏物可以作为影响败血症病程的治疗方式。总的来说，我们报告 CRISPR 介导的内源性Myd88抑制可以有效调节宿主针对 AAV 介导的基因治疗的免疫反应，并影响败血症的病程。使用基于 CRISPR 的合成阻遏物控制Myd88转录水平的能力可能是基于 AAV 的 CRISPR 疗法的有效策略，因为该途径是诱导针对 AAV 血清型的体液免疫的关键节点。