Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona (BIST), Dr. Aiguader 88, 08003, Barcelona, Catalonia, Spain.
Department of Medical Oncology, Bern University Hospital, University of Bern, Inselspital, Switzerland.
BMC Genomics. 2022 May 26;23(1):402. doi: 10.1186/s12864-022-08612-7.
CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays.
CRISPR-Cas9 筛选文库已成为一种强大的工具，可用于鉴定在不同过程中发挥作用的蛋白质编码（pc）和非编码基因。特别是，活性 Cas9 与单个 gRNA 的联合使用已被证明可以通过产生有害的移码来有效削弱 pc 基因的表达。在这里，我们首先证明，同时针对同一基因的两个向导 RNA（配对向导 RNA，pgRNA）协同增强了 CRISPR-Cas9 系统敲除 pc 基因的能力。接下来，我们设计了一个文库，以同时靶向已知在从前 B 细胞向巨噬细胞转分化过程中表达改变的 pc 基因和 lncRNAs。我们表明，该系统能够识别该过程中的已知参与者，并且还预测了 26 个潜在的新参与者，我们从中选择了四个（两个 pc 基因和两个 lncRNAs）进行更深入的表征。我们的结果表明，对于候选 lncRNAs，它们在转分化中的影响可能实际上是由靶向基因座上的增强子区域介导的，而不是由 lncRNA 转录本本身介导的。因此，CRISPR-Cas9 与 pgRNAs 系统的联合使用是一种同时针对 pc 基因和 lncRNAs 进行基因组扰动分析的合适工具。
