CRISPR-Cas9基因编辑技术对细胞内源蛋白进行荧光标记的实验操作

doi:10.16288/j.yczz.22-395

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Hereditas(Beijing) ›› 2023, Vol. 45 ›› Issue (2): 165-175.doi: 10.16288/j.yczz.22-395

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The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing

Zhongsheng Wu^1,²(), Yu Gao^1,²(), Yongtao Du^1,²(), Song Dang¹, Kangmin He^1,²()

1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
2. University of Chinese Academy of Sciences, Beijing 100049, China

Received:2022-12-02 Revised:2023-01-06 Online:2023-02-20 Published:2023-01-09
Contact: He Kangmin E-mail:zswu@genetics.ac.cn;gaoyu@genetics.ac.cn;duyongtao@genetics.ac.cn;kmhe@genetics.ac.cn
Supported by:
the National Natural Science Foundation of China(91957106);the National Natural Science Foundation of China(31970659);the Ministry of Science and Technology of the People’s Republic of China(2021YFA0804802);the Ministry of Science and Technology of the People’s Republic of China(2022YFA1304500)

Abstract

Abstract:

The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.

Key words: CRISPR-Cas9 genome editing, live cell, endogenous protein, fluorescent protein, self-labeling protein tag

Zhongsheng Wu, Yu Gao, Yongtao Du, Song Dang, Kangmin He. The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing[J]. Hereditas(Beijing), 2023, 45(2): 165-175.

Figures/Tables 7

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试剂与耗材名称	品牌	货号
KAPA HiFi HotStart ReadyMix Kit	KAPA Biosystems	KK2631
FastPure Gel DNA Extraction Mini Kit	Vazyme	DC301-01
pEASY-Uni Seamless Cloning and Assembly Kit	TransGen	CU101-03
Quick Ligation? Kit	NEB	M2200L
T4 Polynucleotide Kinase	NEB	M0201S
CutSmart^? Buffer	NEB	B7204S
Sma I	NEB	R0141S
FastDigest^? Bpi I	ThermoFisher Scientific	FD1014
FastAP Thermosensitive Alkaline Phosphatase	ThermoFisher Scientific	EF0652
FastDigest Buffer	ThermoFisher Scientific	B64
GoTaq^? DNA Polymerase	Promega	M300A
TIANamp Genomic DNA Kit	TIANGEN	DP304-02
NucleoBond Xtra Midi Plus	Macherey-Nagel	740412.50
Lipofectamine? 3000	Invitrogen	L3000015
Trypsin-EDTA (0.25%), phenol red	Gibco	25200114
MEM α, Nucleosides, no Phenol Red	Gibco	41061029
Janelia Fluor^? 549 HaloTag^? Ligand	Promega	GA1111
QuickExtract? DNA Extraction Solution	Lucigen	QE09050
LAMP-1 Antibody (H4A3)	Santa Cruz	sc-20011
GAPDH Antibody	Proteintech	10494-1-AP
Cell Strainers	Falcon	352340
Round-Bottom Polystyrene Test Tubes	Falcon	352054
4 Chamber glass bottom dishes	Cellvis	D35C4-20-1.5-N

The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing

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