MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, PR China.
The State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, PR China.
Nucleic Acids Res. 2023 May 22;51(9):e50. doi: 10.1093/nar/gkad198.
Genetic screening based on the clustered regularly interspaced palindromic repeat (CRISPR) system has been indicated to be a powerful tool for identifying regulatory genes or cis-elements. However, when applying CRISPR screens to pinpoint functional elements at particular loci, a large number of guide RNA (gRNA) spacers may be required to achieve saturated coverage. Here, we present a controlled template-dependent elongation (CTDE) method relying on reversible terminators to synthesize gRNA libraries with genomic regions of interest. By applying this approach to H3K4me3 chromatin immunoprecipitation (ChIP)-derived DNA of mammalian cells, mega-sized gRNA libraries were synthesized in a tissue-specific manner, with which we conducted screening experiments to annotate essential sites for cell proliferation. Additionally, we confirmed that an essential site within the intron of LINC00339 regulates its own mRNA and that LINC00339 is a novel regulator of the cell cycle that maintains HepG2 proliferation. The CTDE method has the potential to be automated with high efficiency at low cost, and will be widely used to identify functional elements in mammalian genomes.
基于成簇规律间隔短回文重复序列（CRISPR）系统的遗传筛选已被证明是识别调控基因或顺式元件的有力工具。然而，当将 CRISPR 筛选应用于精确定位特定基因座的功能元件时，可能需要大量的向导 RNA（gRNA）间隔物来实现饱和覆盖。在这里，我们提出了一种依赖于受控模板的延伸（CTDE）方法，该方法利用可逆终止子来合成具有感兴趣基因组区域的 gRNA 文库。通过将这种方法应用于哺乳动物细胞的 H3K4me3 染色质免疫沉淀（ChIP）衍生 DNA，以组织特异性的方式合成了超大尺寸的 gRNA 文库，我们利用这些文库进行了筛选实验，以注释细胞增殖的必需位点。此外，我们还证实，LINC00339 内含子中的一个必需位点调节其自身的 mRNA，并且 LINC00339 是一种新的细胞周期调节剂，能够维持 HepG2 细胞的增殖。CTDE 方法具有高效、低成本的自动化潜力，将广泛用于鉴定哺乳动物基因组中的功能元件。
