Laster Dominique J, Akel Nisreen S, Hendrixson James A, James Alicen, Crawford Julie A, Fu Qiang, Berryhill Stuart B, Thostenson Jeff D, Nookaew Intawat, O'Brien Charles A, Onal Melda
Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Center for Musculoskeletal Disease Research (CMDR), University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
iScience. 2023 Jul 20;26(8):107428. doi: 10.1016/j.isci.2023.107428. eCollection 2023 Aug 18.
Cre-mediated recombination is frequently used for cell type-specific loss of function (LOF) studies. A major limitation of this system is recombination in unwanted cell types. CRISPR interference (CRISPRi) has been used effectively for global LOF in mice. However, cell type-specific CRISPRi, independent of recombination-based systems, has not been reported. To test the feasibility of cell type-specific CRISPRi, we produced two novel knock-in mouse models that achieve gene suppression when used together: one expressing dCas9::KRAB under the control of a cell type-specific promoter and the other expressing a single guide RNA from a safe harbor locus. We then compared the phenotypes of mice in which the same gene was targeted by either CRISPRi or the Cre-loxP system, with cell specificity conferred by regulatory elements in both cases. We demonstrate that CRISPRi is effective for cell type-specific LOF and that it provides improved cell type-specificity compared to the Cre-loxP system.
Cre介导的重组常用于细胞类型特异性功能丧失（LOF）研究。该系统的一个主要局限性是在不需要的细胞类型中发生重组。CRISPR干扰（CRISPRi）已有效地用于小鼠的全局功能丧失研究。然而，尚未报道独立于基于重组的系统的细胞类型特异性CRISPRi。为了测试细胞类型特异性CRISPRi的可行性，我们构建了两种新型的基因敲入小鼠模型，当一起使用时可实现基因抑制：一种在细胞类型特异性启动子的控制下表达dCas9::KRAB，另一种从安全位点表达单个引导RNA。然后，我们比较了通过CRISPRi或Cre-loxP系统靶向相同基因的小鼠的表型，两种情况下均由调控元件赋予细胞特异性。我们证明CRISPRi对细胞类型特异性功能丧失有效，并且与Cre-loxP系统相比，它提供了更好的细胞类型特异性。
