Synthetic Biology Group, Institute for Pharmacy and Biotechnology (IPMB) and Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg 69120, Germany.
Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg 69120, Germany.
Nucleic Acids Res. 2019 Jul 26;47(13):e75. doi: 10.1093/nar/gkz271.
The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.
CRISPR-Cas 技术的快速发展使得针对遗传疾病的个性化和靶向治疗成为可能。为了使基于 CRISPR 的治疗方法精确和安全，人们非常希望有一种策略可以将 Cas(9)的活性限制在选定的细胞和组织中。在这里，我们开发了一种基于 miRNA 调控抗 CRISPR（Acr）蛋白表达的细胞类型特异性 Cas-ON 开关。我们将 miR-122 或 miR-1 的靶位点插入 Acr 转基因的 3'UTR 中，miR-122 或 miR-1 分别在肝脏和心肌细胞中特异性丰富。共表达这些与 Cas9 和 sgRNA 导致 Acr 敲低，并仅在肝细胞或心肌细胞中释放 Cas9 活性，而 Cas9 在非靶细胞中被有效抑制。我们使用依赖 miR 的 AcrIIA4 与不同的酿脓链球菌（Spy）Cas9 变体（全长 Cas9、分裂 Cas9、dCas9-VP64）结合，证明了对基因组编辑和基因激活的控制。最后，为了展示其模块化，我们将我们的 Cas-ON 系统适应了更小且更具特异性的脑膜炎奈瑟菌（Nme）Cas9 同源物及其同源抑制剂 AcrIIC1 和 AcrIIC3。我们的 Cas-ON 开关应该可以促进任何已知有有效抗 CRISPR 蛋白的 CRISPR-Cas 同源物的细胞特异性活性。
