Center for Molecular and Cellular Bioengineering (CMCB), Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Dresden, Germany.
Center for Molecular and Cellular Bioengineering (CMCB), DRESDEN-Concept Genome Center, Technische Universität Dresden, Dresden, Germany.
Nat Commun. 2021 Feb 18;12(1):1125. doi: 10.1038/s41467-021-21427-6.
Conditional gene inactivation is a powerful tool to determine gene function when constitutive mutations result in detrimental effects. The most commonly used technique to achieve conditional gene inactivation employs the Cre/loxP system and its ability to delete DNA sequences flanked by two loxP sites. However, targeting a gene with two loxP sites is time and labor consuming. Here, we show Cre-Controlled CRISPR (3C) mutagenesis to circumvent these issues. 3C relies on gRNA and Cre-dependent Cas9-GFP expression from the same transgene. Exogenous or transgenic supply of Cre results in Cas9-GFP expression and subsequent mutagenesis of the gene of interest. The recombined cells become fluorescently visible enabling their isolation and subjection to various omics techniques. Hence, 3C mutagenesis provides a valuable alternative to the production of loxP-flanked alleles. It might even enable the conditional inactivation of multiple genes simultaneously and should be applicable to other model organisms amenable to single integration transgenesis.
条件性基因失活是一种强大的工具，可用于确定基因功能，当组成性突变产生有害影响时。最常用的实现条件性基因失活的技术采用 Cre/loxP 系统及其删除两侧带有两个 loxP 位点的 DNA 序列的能力。然而，靶向带有两个 loxP 位点的基因既费时又费力。在这里，我们展示了 Cre 控制的 CRISPR（3C）诱变，以规避这些问题。3C 依赖于同一转基因中 gRNA 和 Cre 依赖性 Cas9-GFP 的表达。外源性或转基因 Cre 的供应导致 Cas9-GFP 的表达和随后的感兴趣基因的突变。重组细胞变得荧光可见，从而能够分离它们并进行各种组学技术。因此，3C 诱变提供了一种替代产生 loxP 侧翼等位基因的有价值的方法。它甚至可以实现多个基因的同时条件性失活，并且应该适用于其他可进行单一整合转基因的模式生物。
