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用CRISPR/Cas9对CAR-T细胞进行多重基因编辑（一）

2020.5.18

作者：

Xiaojuan Liu1, *, Yongping Zhang2, *, Chen Cheng1, 3, *, Albert W Cheng4, Xingying Zhang1, 5, Na Li1, Changqing Xia2, 6, Xiaofei Wei7, Xiang Liu1, Haoyi Wang1, 5, 8

单位：

1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China;

2Department of Hematology, Xuanwu Hospital, Capital Medical University, Beijing, China;

3Graduate School, University of Science and Technology of China, Hefei, China;

4The Jackson Laboratory for Genome Medicine, Farmington, CN, USA;

5University of Chinese Academy of Sciences;

6Department of Pathology, Immunology and Laboratory Medicine, University of Florida, FL, USA;

7Beijing Cord Blood Bank, Beijing, China;

8The Jackson Laboratory, Bar Harbor, ME, USA

实验材料：

名称	用途	来源
Fresh umbilical cord blood (UCB) units	分离脐带血T细胞	Beijing Cord Blood Bank (Beijing, China)
Histopaque-1077	密度梯度离心	(Sigma-Aldrich)
EasySep human T cell enrichment kit	富集T细胞	(Stemcell Technologies)
anti-CD3/anti-CD28 Dynabeads	激活T细胞	(ThermoFisher Scientific)
X-vivo15 medium	培养T细胞	(Lonza)
heat-inactivated fetal bovine serum	血清，培养T细胞和各种细胞系	(ThermoFisher Scientific)
L-glutamine	谷氨酰胺，培养T细胞和各种细胞系	(ThermoFisher Scientific)
sodium pyruvate	丙酮酸钠，培养T细胞和各种细胞系	(ThermoFisher Scientific)
recombinant human IL-2	白介素2，激活T细胞	(ThermoFisher Scientific)
Trypan blue	台盼蓝，细胞计数	(ThermoFisher Scientific)
Raji	淋巴瘤细胞系	(Burkitt’s lymphoma cell line, ATCC-CCL86)
Daudi	B细胞淋巴瘤细胞系	(B lymphoblast cell line, ATCC-CCL213)
K562 cells	人红白血病细胞系	(human erythroleukemic cell line, ATCC-CCL243)
48-well plates		(Corning)
ﬁreﬂy luciferase lentiviral particles	荧光素酶慢病毒	Genechem (Shanghai, China)
MoFlo XDP	K562细胞分选	Beckman Coulter Inc
RPMI1640 medium	Raji/Daudi/K562细胞系培养	(ThermoFisher Scientific)
293T	生产病毒	(ATCC-CRL3216)
DMEM	培养293T细胞，加10%血清	(ThermoFisher Scientific)
pMD2.G, psPAX2	包装病毒的质粒	(addgen)
Lipofectamine3000	转染293T包装病毒	(ThermoFish Scientific)
ultracentrifugation	超速离心分离病毒	(Merck Millipore)
pX330 plasmid	sgRNA编码质粒	(Addgene plasmid #4223)
MEGAshortscript T7 kit	体外转录得到sgRNA	(ThermoFisher Scientific)
MEGAclear columns	纯化RNA	(ThermoFisher Scientific)
Cas9 protein	Cas9蛋白	(ThermoFisher Scientific)
4D-Nucleofector System N	4D核转	(Lonza)
P3 Primary Cell 4D Nucleofector X Kit	核转试剂盒，转T细胞	(Lonza) V4XP-3024
DPBS	DPBS	ThermoFisher Scientific
CytoFLEX	流式检测	(Beckman Coulter Inc)
TCR α/β-PE/Cy7	流式抗体	(IP26, Biolegend),
TCRα/β-PE	流式抗体	(IP26, Biolegend),
β2-microglobulin (B2M)-PE	流式抗体	(2M2, Biolegend),
β2-microglobulin (B2M)-FITC	流式抗体	(2M2, Biolegend),
CD279(PD-1)-APC	流式抗体	(EH12.2H7, Biolegend),
CD279(PD-1)-PE	流式抗体	(EH12.2H7, Biolegend),
HLA-A2-FITC	流式抗体	(BB7.2, BD Pharmingen)
surveyor mutation detection kit	突变检测试剂盒	(Integrated DNA Technologies, Inc)
pEASY Blunt Cloning Kit	平末端克隆	(Transgen Biotech)
EasySep PE selection kit	分离CAR-T细胞	(Stemcell Technologies)
ELISA	检测白介素和干扰素γ	(Biolegend)
Celltrace Violet	细胞坏死检测	(ThermoFisher Scientific)
FITC-AnnexinV and 7-AAD	染色后做流式	(Biolegend)
6-12-week-old NOD-Prkdcscid Il2rgnull (NPG) mice	小鼠	(VITALSTAR, Beijing, China)
matrigel matrix	混合肿瘤细胞，做活体注射	(Corning)
Xenogen IVIS Imaging System	活体成像系统	(Perkin Elmer Life Sciences)
d-luciferin	活体成像底物	(Perkin Elmer Life Sciences)

实验方法：

从脐带血中分离并扩增T细胞 Isolation and expansion of T cells from UCB units

Fresh umbilical cord blood (UCB) units were obtained from healthy volunteer donors who had provided informed consent from the Beijing Cord Blood Bank (Beijing, China), and mononuclear cells were separated using density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich). T cells were isolated using the EasySep human T cell enrichment kit (Stemcell Technologies), activated and expanded with anti-CD3/anti-CD28 Dynabeads (ThermoFisher Scientific) at a bead to T cell ratio of 1:1 according to the manufacturer's instructions. UCB-derived T cells were cultured in X-vivo15 medium (Lonza) supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 2mM L-glutamine, and 1mM sodium pyruvate in the presence of 300 IU/mL recombinant human IL-2 (all from ThermoFisher Scientific). Viable cells were enumerated using Trypan blue (ThermoFisher Scientific) exclusion. All cells were grown at 37 °C in a 5% CO2 atmosphere.

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用CRISPR/Cas9对CAR-T细胞进行多重基因编辑（一）

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