Amalie Dyrelund Broksø, Louise Bendixen, Simon Fammé, Kasper Mikkelsen, Trine Ilsø Jensen, Rasmus O Bak
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引用次数: 0
摘要
基于 CRISPR/Cas 的转录激活(CRISPRa)和干扰(CRISPRi)通过将转录调节剂招募或融合到核酶缺陷 Cas(dCas)上,实现了瞬时可编程基因调控。在这里,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。我们利用金黄色葡萄球菌和化脓性链球菌的 dCas9 进行正交转录调节,在上调一组基因的同时下调另一组基因。我们还将正交转录调控与 Cas12a(Acidaminococcus;AsCas12a)核糖核蛋白(RNP)递送基因敲除相结合,建立了三模式基因工程。为了简化三模式工程,我们探索了实施截短 sgRNA 的最佳参数,以便利用 SpCas9 进行基因敲除和 CRISPRa。我们发现 Cas9 蛋白与 sgRNA 的比例对于避免 sgRNA 交叉复合物以及平衡基因敲除和激活效率至关重要。我们在原代人类 T 细胞中展示了三模式基因工程的高效率,同时保持了 T 细胞的基本健康和功能。这项研究凸显了使用多种 CRISPR/Cas 系统进行复杂基因工程的多功能性和潜力,只需一个简单的步骤,就能实现瞬时转录组调控和永久性 DNA 改变。我们相信,这种精心设计的工程可用于再生医学和疗法,以促进更复杂的治疗。
Orthogonal transcriptional modulation and gene editing using multiple CRISPR-Cas systems.
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.
期刊介绍:
Molecular Therapy is the leading journal for research in gene transfer, vector development, stem cell manipulation, and therapeutic interventions. It covers a broad spectrum of topics including genetic and acquired disease correction, vaccine development, pre-clinical validation, safety/efficacy studies, and clinical trials. With a focus on advancing genetics, medicine, and biotechnology, Molecular Therapy publishes peer-reviewed research, reviews, and commentaries to showcase the latest advancements in the field. With an impressive impact factor of 12.4 in 2022, it continues to attract top-tier contributions.