Broksø Amalie Dyrelund, Bendixen Louise, Fammé Simon, Mikkelsen Kasper, Jensen Trine Ilsø, Bak Rasmus O
Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
Mol Ther. 2025 Jan 8;33(1):71-89. doi: 10.1016/j.ymthe.2024.11.024. Epub 2024 Nov 19.
CRISPR-Cas-based transcriptional activation (CRISPRa) and interference (CRISPRi) enable transient programmable gene regulation by recruitment or fusion of transcriptional regulators to nuclease-deficient Cas (dCas). Here, we expand on the emerging area of transcriptional engineering and RNA delivery by benchmarking combinations of RNA-delivered dCas and transcriptional modulators. We utilize dCas9 from Staphylococcus aureus and Streptococcus pyogenes for orthogonal transcriptional modulation to upregulate one set of genes while downregulating another. We also establish trimodal genetic engineering by combining orthogonal transcriptional regulation with gene knockout by Cas12a (Acidaminococcus; AsCas12a) ribonucleoprotein delivery. To simplify trimodal engineering, we explore optimal parameters for implementing truncated single guide RNAs (sgRNAs) to make use of SpCas9 for knockout and CRISPRa. We find the Cas9 protein/sgRNA ratio to be crucial for avoiding sgRNA cross-complexation and for balancing knockout and activation efficiencies. We demonstrate high efficiencies of trimodal genetic engineering in primary human T cells while preserving basic T cell health and functionality. This study highlights the versatility and potential of complex genetic engineering using multiple CRISPR-Cas systems in a simple one-step process yielding transient transcriptome modulation and permanent DNA changes. We believe such elaborate engineering can be implemented in regenerative medicine and therapies to facilitate more sophisticated treatments.
基于CRISPR-Cas的转录激活（CRISPRa）和干扰（CRISPRi）通过将转录调节因子招募或融合到核酸酶缺陷型Cas（dCas）来实现瞬时可编程基因调控。在此，我们通过对RNA递送的dCas和转录调节剂的组合进行基准测试，拓展了转录工程和RNA递送这一新兴领域。我们利用来自金黄色葡萄球菌和化脓性链球菌的dCas9进行正交转录调控，以上调一组基因同时下调另一组基因。我们还通过将正交转录调控与Cas12a（嗜氨基酸球菌；AsCas12a）核糖核蛋白递送介导的基因敲除相结合，建立了三模态基因工程。为了简化三模态工程，我们探索了实施截短的单向导RNA（sgRNA）以利用SpCas9进行敲除和CRISPRa的最佳参数。我们发现Cas9蛋白与sgRNA的比例对于避免sgRNA交叉复合以及平衡敲除和激活效率至关重要。我们在原代人T细胞中展示了三模态基因工程的高效性，同时保留了基本的T细胞健康和功能。这项研究突出了在一个简单的一步过程中使用多种CRISPR-Cas系统进行复杂基因工程的多功能性和潜力，该过程可产生瞬时转录组调节和永久性DNA改变。我们相信这种精细的工程可应用于再生医学和治疗中，以促进更复杂的治疗。
