State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
Key Laboratory for Plague Prevention and Control of Qinghai Province 2017-ZJ-Y15, Institute for Endemic Disease Prevention and Control of Qinghai Province, Xining, China.
Appl Environ Microbiol. 2019 May 30;85(12). doi: 10.1128/AEM.00097-19. Print 2019 Jun 15.
Many genes in the bacterial pathogen , the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system using Cas9-derived dCas9 for gene knockdown in Multiple genes harbored on either the chromosome or plasmids of were efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of (responsible for biofilm formation) or (encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance in phenotypic assays. Furthermore, silencing of the virulence-associated genes or using this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported for and null mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in , providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening of genes with unknown functions. is a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, many genes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported in Here, we systematically optimized the CRISPRi approach for use in , which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected by assays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach in and highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.
许多细菌病原体（导致三次鼠疫大流行的病原体）的基因仍未被描述，这极大地阻碍了鼠疫预防和控制措施的发展。成簇规律间隔短回文重复干扰（CRISPRi）已被证明是一种在模型细菌中基因敲低的有效工具。在该系统中，一种无催化活性的 Cas9（dCas9）和一个小向导 RNA（sgRNA）形成复合物，通过碱基配对与特定的 DNA 靶标结合，从而阻碍 RNA 聚合酶的结合并导致靶基因的抑制。在这里，我们引入了一种优化的 CRISPRi 系统，使用 Cas9 衍生的 dCas9 在严格的脱水四环素诱导下，在 染色体或质粒上的多个基因都能有效地被敲低（高达 380 倍）。用这种 CRISPRi 方法敲低 （负责生物膜形成）或 （编码冷休克蛋白）导致生物膜形成大大减少或在表型测定中冷耐受性受损。此外，用这种 CRISPRi 系统沉默毒力相关基因 或 导致在 HeLa 细胞和小鼠中的毒力减弱，与之前报道的 和 缺失突变体相似。总之，我们的结果证实，这种优化的 CRISPRi 系统可以可逆且有效地抑制 中的靶基因表达，为常规基因敲低技术提供了替代方案，也是筛选具有未知功能的 基因的高通量表型筛选策略。是一种致命的病原体，在历史上导致了数百万人死亡。它也因其作为生物武器或生物恐怖主义制剂的潜在用途而受到广泛关注，针对这种病原体急需新的疫苗。然而，许多 基因仍未被描述，这极大地阻碍了鼠疫预防和控制措施的发展。成簇规律间隔短回文重复干扰（CRISPRi）已在多种细菌的功能基因组研究中成功应用，但在 中尚未报道过这种遗传工具。在这里，我们系统地优化了 CRISPRi 方法在 中的应用，最终以可逆的方式高效地抑制了靶基因的表达。用这种方法敲低功能基因产生的表型可以通过 测定、细胞感染测定和小鼠感染实验很容易地检测到。这是在 中使用 CRISPRi 方法的报告，并强调了该方法在该病原体高通量功能基因组学研究中的潜在用途。
