建立干擾型CRISPR篩選平台以研究替代型活化巨噬細胞

Abstract

巨噬細胞是先天免疫系統中的一種白血球，可以根據微環境中的刺激極化為促炎性的M1巨噬細胞或抗炎性的M2巨噬細胞。M1巨噬細胞在清除病原體方面發揮著至關重要的作用，而M2巨噬細胞主要參與組織修復。腫瘤微環境中存在許多巨噬細胞，它們對腫瘤的進展具有顯著的影響。值得注意的是，腫瘤微環境中的M2巨噬細胞促進腫瘤生長，並與癌症的不良預後有關。然而，M2巨噬細胞極化的詳細機制仍然大部分未知。我們最終的目標是建立一個利用CRISPR干擾（CRISPR interference，CRISPRi）的基因組範圍篩選系統，以識別參與M2巨噬細胞極化的新因子。然而，由於巨噬細胞中轉導和轉染效率較低，本研究專注於解決建立CRISPRi篩選系統時，主要遇到的兩個困難：1）在巨噬細胞系中穩定表達功能性的dCas9-KRAB蛋白；2）在巨噬細胞系中轉導足夠多樣性的sgRNA。在這裡，我們首先在NIH-3T3和RAW264.7細胞中建立了dCas9-KRAB介導的基因抑制系統，然後檢測了之前建立的dCas9-KRAB在骨髓細胞中的功能性（immortalized bone marrow cell，iBM）。此外，我們做了一個小型sgRNA庫，其中包含15個不同的sgRNA，並利用它檢測了iBM中sgRNA的多樣性和均勻性。我們的研究結果使得在iBM中建立CRISPRi篩選系統有進一步的進展。

Macrophages, a type of leukocytes in innate immune system, can be polarized to pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages according to the stimuli in the microenvironment. M1 macrophages play a vital role in pathogen clearance, while M2 macrophages predominantly participate in tissue repair. Macrophages are enriched within the tumor microenvironment and significantly influence tumor progression. Notably, M2 macrophages in tumor microenvironment promote tumor growth, and have been implicated in poor prognosis of cancer. However, the detailed mechanism of the M2 macrophage polarization are still largely unknown. Though our eventual goal is to establish a genome-wide screening system using CRISPR-interference (CRISPRi) to identify novel factors that participate M2 macrophage polarization. However, due to the poor transduction and transfection efficiency in macrophages, this study focuses on addressing two major difficulties that are encountered in establishing the CRISPRi screening system, 1) stably expressing functional dCas9-KRAB protein in macrophage cell line. 2) Transduce sufficient diversity of sgRNAs in macrophage cell line. Here, we first established the dCas9-KRAB mediated gene knockdown system in both NIH-3T3 and RAW264.7 cells, and followed by examining the functionality of the previously established dCas9-KRAB in immortalized bone marrow cells (iBM). Furthermore, the diversity and evenness of integrated sgRNAs in iBM was examined by a newly generated mini-library containing 15 different sgRNAs. Our results provide a further advance in establishing CRISPRi screening system in iBM.