Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia 2371, Cyprus.
Cyprus School of Molecular Medicine, Nicosia 2371, Cyprus.
Int J Mol Sci. 2022 Jan 19;23(3):1082. doi: 10.3390/ijms23031082.
Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin repressor in erythroid cells by tagging the 3' untranslated region (UTR) of with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongside double- or single-stranded oligodeoxynucleotides for, respectively, non-homologous-end-joining (NHEJ)- or homology-directed-repair (HDR)-mediated MRS insertion. NHEJ-based tagging was imprecise and inefficient (≤6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (≤18%), but toxic for longer donors encoding concatenated and thus potentially more efficient MRS tags. Isolation of clones for robust HEK293T cells tagged with a homozygous quadruple MRS resulted in 25% spontaneous reduction in BCL11A and up to 36% reduction after transfection with an miR-451a mimic. Isolation of clones for human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells tagged with single or double MRS allowed detection of albeit weak γ-globin induction. Our study demonstrates suitability of TAMED for physiologically relevant modulation of gene expression and its unsuitability for therapeutic application in its current form.
分子疗法和功能研究极大地受益于遗传干预的时空精度。因此，我们构思并探索了标记激活 microRNA（miRNA）介导的内源性失活（TAMED）作为一种研究工具和潜在的谱系特异性治疗方法。为了验证原理，我们旨在通过标记 3'非翻译区（UTR）与丰富的红细胞 microRNA miR-451a 的 miRNA 识别位点（MRS）来失活红细胞中的 γ-珠蛋白抑制剂。为此，我们采用 CRISPR/Cas9 核糖核蛋白（RNP）颗粒的核转染以及双链或单链寡脱氧核苷酸，分别用于非同源末端连接（NHEJ）或同源定向修复（HDR）介导的 MRS 插入。基于 NHEJ 的标记是不精确和低效的（≤6%），并且均匀地产生了敲入和包含插入/缺失的 MRS 标记，而基于 HDR 的标记则更有效（≤18%），但对于较长的供体编码串联的 MRS 标记则具有毒性，因此可能更有效。对具有同源四倍 MRS 标记的稳定 HEK293T 细胞进行克隆分离，导致 BCL11A 自发减少 25%，转染 miR-451a 模拟物后减少 36%。对单或双 MRS 标记的人脐带血衍生红细胞祖细胞-2（HUDEP-2）细胞进行克隆分离，允许检测到尽管较弱的 γ-珠蛋白诱导。我们的研究表明 TAMED 适用于生理相关的基因表达调节，但其在当前形式下不适合治疗应用。
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