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Abstract
Introduction
Protocol
Results
Discussion
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Acknowledgements
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Immunology and Infection

基因敲击由CRISPR/Cas9和细胞排序在巨噬细胞和T细胞线

Published: November 13, 2021
doi:
10.3791/62328
, , , , , , , , ,
1The Laboratory of Genetic Regulators in the Immune System, School of Laboratory Medicine,Xinxiang Medical University, 2Centre d’Immunologie de Marseille-Luminy,Aix Marseille Université

Summary

该协议使用荧光记者和细胞分拣来简化巨噬细胞和T细胞系的敲击实验。两个质粒用于这些简化的敲击实验，即CRISPR/Cas9-和DsRed2表达质粒和同源重组供体质粒表达EBFP2，这是永久整合在 Rosa26 点在免疫细胞。

Abstract

免疫系统的功能基因组学研究需要基因操作，包括删除目标基因和在感兴趣的蛋白质中添加元素。细胞系模型中基因功能的识别对于基因的发现和细胞内在机制的探索具有重要意义。然而，使用CRISPR/Cas9介质敲击的免疫细胞（如T细胞和巨噬细胞系）的基因操作是困难的，因为这些细胞的转染效率低，尤其是在静止状态下。为了改变免疫细胞中的基因，耐药性选择和病毒载体通常用于丰富表达CRIPSR/Cas9系统的细胞，这不可避免地导致细胞的不良干预。在之前的研究中，我们设计了双荧光记者耦合到CRISPR/Cas9，在电透后短暂表达。这种技术解决方案可导致免疫细胞中快速基因缺失：然而，基因敲击免疫细胞，如T细胞和巨噬细胞，而不使用耐药性选择或病毒载体是更具挑战性的。在本文中，我们表明，通过使用细胞分拣来帮助选择暂时表达CRISPR/Cas9结构的目标 Rosa26 细胞群结合供体质粒，基因敲击可以在T细胞和巨噬细胞中实现，而无需耐药性浓缩。例如，我们通过进行敲击实验，在RAW264.7巨噬细胞中展示如何表达人类ACE2，SARS-Cov-2的受体，它负责当前Covid-19大流行。这种基因敲击细胞可以广泛用于机械研究。

Introduction

免疫细胞对防御病原体至关重要。先天免疫和适应性免疫是需要清除感染剂和维持组织平衡1，2。细胞系模型是了解哺乳动物免疫系统分子基本原理的重要工具：它们用于体外功能测定，如模拟人体T细胞活化，以及确定遗传因素在激活或抑制免疫反应中的功能3，4。需要注意的是，哺乳动物的免疫系统是极其异质的，同样重要的是，大量的分子控制着特定细胞类型5、6的分化、迁移和功能。

聚类定期间歇性短白细胞重复（CRISPR）/Cas9基因组编辑工具允许特定细胞类型的基因操作，从而促进基因的功能注释以精确的方式7，8。一些已发表的协议描述了CRISPR/Cas9的交付形式Cas9导引RNA复合物称为核糖核蛋白（RNP）在HIK293细胞，Jurkat细胞系，原发T细胞9，10，巨噬细胞11，12，13，干细胞14，和其他15，16。在这些协议中，基因标记通常是通过将荧光标签与内源性蛋白质17、18融合来实现的。然而，很少尝试使用双荧光记者，这是兼容的单细胞排序，以促进敲击实验19，20，特别是在免疫细胞。

旨在理解免疫细胞中新基因因子功能的深入机械分析通常需要细胞类型特定地删除基因、基因拯救实验以及理想地识别其相互作用物。尽管在免疫细胞中优化基因缺失的方法已经公布9，15，21，但关于引入具有多功能的敲击等位基因来理解免疫反应的方法却少得多。因此，在这个协议中，我们旨在详细描述一个高效和高度可重复的协议，以表达一个蛋白质的兴趣在安全港点Rosa26在人类和穆林免疫细胞线。我们设计了一个双色报告系统，用于丰富用质粒表示CRISPR/Cas9（DsRed2）和重组DNA模板（EBFP2）的细胞，这些模板可以通过细胞分拣进行分离。根据这个协议，我们获得了人类T细胞系Jurkat和穆林巨噬细胞RAW264.7的多个敲击线，用于对研究不善的蛋白质进行功能分析。

例如，我们在此协议中展示如何获得可敲击 RAW264.7 巨噬细胞，稳定地表达人类ACE2（SARS-Cov-2的受体）22。由于先天免疫细胞参与Covid-1923、24和人类ACE2的发病机制，被认为是病毒在复制前进入细胞所需的主要受体，具有人类ACE2的微噬细胞可以作为巨噬细胞内病毒增殖的机械研究的有用工具。同时，我们还介绍了一个基因在人类ROS26位点敲击来表达RASGRP1蛋白的例子，该蛋白在其氨基终点与亲和力双链球菌标签（OST）融合在一起。T细胞是免疫疗法中的关键靶细胞，越来越多的研究集中在对癌症的反应上。由于Rasgrp1是T细胞受体下游的关键信号分子，其相互作用因素27日没有得到很好的阐明，OST-RASGRP1的敲击模型为识别调节T细胞对肿瘤和感染反应的相互作用者奠定了基础。综合起来，这些工具可用于Covid-19的研究和发现与Rasgrp1相互作用的新分子。

Protocol

1. 以 罗莎26 蝗虫为目标的 sgRNA 的设计和普拉斯米德构造 围绕所需插入站点的设计指南 RNA 确保鼠标Rosa26（以下简称mRosa26）的插入点位于mRosa26的第一个内电路：这个网站已经用于以前的研究28，29。对于人体细胞的敲击实验，确保插入位点位于人类ROSA26位点（以下简称hROSA26）?…

Representative Results

按照上述协议，使用木乃伊RAW264.7巨噬细胞在 mRosa26 靶点进行敲击实验，我们设计了一个靶向载体来表达人类ACE2，SARS-Cov-2病毒的受体（图2A）。使用类似的设计，我们生成了人类Jurkat T细胞与OST标记RASGRP1融合蛋白（图2C）的敲击。在转染三个质粒后，其中两个用于表示 CRISPR/Cas9 （DsRed2; pdsR-mR26-sg1 和 pDsR-mR-sg2 用于 mRosa26 敲击; pdsR-hR26-sg1 和 …

Discussion

在我们的实验中，我们演示了如何使用人类 Jurkat T 细胞和 Murine RAW264.7 巨噬细胞进行免疫细胞的敲击编辑，从结构设计到敲击细胞筛选和验证。T细胞和巨噬细胞系都耐转染36，37：然而，CRISPR/Cas9交付效率低下的问题可以通过荧光记者的辅助和细胞分拣来克服。本协议适用于基因抢救实验和蛋白质-蛋白质相互作用实验，但不能应用于调控DNA序列的研究，…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢新乡医科大学的流细胞学核心设施。国家自然科学基金向LZ、81471595和32070898提供81601360支持了这种技术的发展。这项工作也得到了河南省教委第21号IRTSTHN030基金会的支持。

Materials

Amersham Imager 600 Ge Healthcare imaging of chemiluminescence
Ampicillin, sodium salt MP Biomedicals 194526
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling Technology 7074 at 1/5000 dilution
Anti-RasGRP1 antibody, clone 10.1 Merck MABS146 1.0 μg/mL of working concentration
AscI New England BioLabs R0558S
β-Actin (D6A8) Rabbit mAb Cell Signaling Technology 8457 at 1/1000 dilution
BamHI-HF New England BioLabs R3136S
BbsI-HF New England BioLabs R3539S
Cellometer Mini Automated Cell Counter Nexcelom Bioscience
E.coli DH5α Competent Cells Takara 9057
DMSO (Dimethyl Sulfoxide) MP Biomedicals 196055
DNeasy Blood & Tissue Kits Qiagen 69506 cell culture reagent
DPBS (10X), no calcium, no magnesium ThermoFisher Scientific 14200075
Dulbecco's Modified Eagle Medium (DMEM) with high glucose HyClone SH30022.01
EcoRI-HF New England BioLabs R3101S
FACSAria™ Fusion BD Biosciences equipped with biosafety cabinet
FACS Canto flow cytometer BD Biosciences
Falcon 5 ml polystyrene round bottom test tube BD Biosciences 352003
Fetal bovine serum (FBS) ThermoFisher Scientific 10099141
FlowJo version 10.7 BD Biosciences
GAPDH (D16H11) XP Rabbit mAb Cell Signaling Technology 5174 at 1/1000 dilution
Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP ThermoFisher Scientific 31430 at 1/5000 dilution
Immobilon ECL Ultra Western HRP Substrate Millipore WBKLS0500
Immobilon-PSQ PVDF Membrane Millipore ISEQ00010
Jurkat ATCC TIB-152 https://www.atcc.org/
Kanamycin sulfate MP Biomedicals 194531
LB agar powder ThermoFisher Scientific 22700041
Multi-channel Pipette (30-300 μL) Eppendorf, or similar
Neon Transfection System ThermoFisher Scientific MPK5000
Neon Transfection System, 10 μL kit ThermoFisher Scientific MPK1096
Nunc 15 mL Conical Sterile Centrifuge Tubes ThermoFisher Scientific 339651
OneTaq® Hot Start Quick-Load® 2X Master Mix New England BioLabs (M0489) for high GC% template
PageRuler Prestained Protein Ladder, 10 to 180 kDa ThermoFisher Scientific 26616
Pipette tip 0.1-20µl Eppendorf, or similar 0030 075.005
Pipette tip 2-200µl Eppendorf, or similar 0030 075.021
Pipette tip 50-1000µl Eppendorf, or similar 0030 075.064
Plasmid Maxi Kit Qiagen 12163
pX458-DsRed2 Addgene 112219
QIAquick PCR Purification Kit Qiagen 28104 purify plasmid from restriction digestion
Q5 Hot Start High-Fidelity 2X Master Mix New England BioLabs M0494S
RAW264.7 ATCC TIB-71 https://www.atcc.org/
Recombinant Anti-ACE2 antibody [EPR4435(2)] Abcam ab108252 at 1/1000 dilution
RPMI 1640 Medium HyClone SH30027.01
Strep-Tactin Sepharose beads IBA Lifesciences 2-1201-010
Penicillin-Streptomycin ThermoFisher Scientific 15140122
SYTOX™ Red Dead Cell Stain, for 633 or 635 nm excitation ThermoFisher Scientific S34859
T4 DNA ligase New England BioLabs M0202S
T4 Polynucleotide Kinase New England BioLabs M0201S
Trypan Blue Solution, 0.4% ThermoFisher Scientific 15250061
Trypsin-EDTA solution (0.25%), with phenol red ThermoFisher Scientific 25200056
ZOE Fluorescent Cell Imager Bio-Rad
1.5 mL microtubes, PCR-clean Eppendorf, or similar 0030 125.215
24-well Clear TC-treated Multiple Well Plates Corning 3524
96-well Clear Flat Bottom Polystyrene TC-treated Microplates Corning 3599
96-well Clear Round Bottom TC-treated Microplate Corning 3799
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Tags

CRISPR/Cas9Gene Knock-inMacrophageT Cell LinesROSA26 LocusSgRNA DesignHomologous RecombinationTargeting VectorFluorescent ReporterCell Sorting

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Zhang, L., Huang, R., Lu, L., Fu, R., Guo, G., Gu, Y., Liu, Z., He, L., Malissen, M., Liang, Y. Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines. J. Vis. Exp. (177), e62328, doi:10.3791/62328 (2021).
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