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.

中文翻译：

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使用多个 CRISPR-Cas 系统进行正交转录调控和基因编辑


基于 CRISPR-Cas 的转录激活 （CRISPRa） 和干扰 （CRISPRi） 通过将转录调节因子募集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，扩展了转录工程和 RNA 递送的新兴领域。我们利用来自金黄色葡萄球菌和化脓性链球菌的 dCas9 进行正交转录调节，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（酸性球菌;AsCas12a） 核糖核蛋白递送。为了简化三峰工程，我们探索了实现截短单向导 RNA （sgRNA） 的最佳参数，以利用 SpCas9 进行敲除和 CRISPRa。我们发现 Cas9 蛋白/sgRNA 比率对于避免 sgRNA 交叉复合以及平衡敲除和激活效率至关重要。我们在原代人类 T 细胞中展示了三峰基因工程的高效性，同时保留了基本的 T 细胞健康和功能。本研究强调了复杂基因工程的多功能性和潜力，使用多个 CRISPR-Cas 系统在一个简单的一步过程中产生瞬时转录组调节和永久性 DNA 变化。我们相信这种精心设计的工程可以在再生医学和疗法中实施，以促进更复杂的治疗。