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

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

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

吴仲胜^1,²(), 高誉^1,²(), 杜勇涛^1,²(), 党颂¹, 何康敏^1,²()

1.中国科学院遗传与发育生物学研究所，分子发育生物学国家重点实验室，北京 100101
2.中国科学院大学，北京 100049

收稿日期:2022-12-02 修回日期:2023-01-06 出版日期:2023-02-20 发布日期:2023-01-09
通讯作者: 何康敏 E-mail:zswu@genetics.ac.cn;gaoyu@genetics.ac.cn;duyongtao@genetics.ac.cn;kmhe@genetics.ac.cn
作者简介:吴仲胜，博士研究生，专业方向：细胞脂质信号转导。E-mail: zswu@genetics.ac.cn
基金资助:
国家自然科学基金项目(91957106);国家自然科学基金项目(31970659);国家重点研发计划(2021YFA0804802);国家重点研发计划(2022YFA1304500)

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)

摘要/Abstract

摘要：

CRISPR-Cas9是目前广泛应用的基因编辑技术，可对目的基因进行高效精准编辑，快速实现目的基因的敲除或敲入。Cas9蛋白在sgRNA引导下对靶序列进行剪切并造成DNA双链断裂，在与剪切位点两端同源的DNA模板序列存在时，可通过同源重组修复方式引入外源序列，实现荧光蛋白或其他标签在基因组上的精准敲入，进而实现对内源蛋白进行荧光标签的融合标记。通过基因编辑技术对内源目的蛋白进行标记，可避免由于过表达造成蛋白质定位、动力学或功能等的潜在影响，可显著提升细胞成像实验的稳定性和可重复性。本文重点介绍了利用CRISPR-Cas9基因编辑系统对目的蛋白进行荧光蛋白或自标记蛋白标签标记的方法与操作流程，为构建内源蛋白荧光标记的哺乳动物细胞系提供参考。

关键词: CRISPR-Cas9基因编辑, 活细胞, 内源蛋白, 荧光蛋白, 自标记蛋白标签

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

吴仲胜, 高誉, 杜勇涛, 党颂, 何康敏. 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[J]. 遗传, 2023, 45(2): 165-175.

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.

图/表 7

图1

图2

表1

图3

图4

图5

图6

参考文献 39

[1]	Schneider AFL, Hackenberger CPR. Fluorescent labelling in living cells. Curr Opin Biotechnol, 2017, 48: 61-68. doi: 10.1016/j.copbio.2017.03.012
[2]	Crivat G, Taraska JW. Imaging proteins inside cells with fluorescent tags. Trends Biotechnol, 2012, 30(1): 8-16. doi: 10.1016/j.tibtech.2011.08.002 pmid: 21924508
[3]	Cranfill PJ, Sell BR, Baird MA, Allen JR, Lavagnino Z, de Gruiter HM, Kremers GJ, Davidson MW, Ustione A, Piston DW. Quantitative assessment of fluorescent proteins. Nat Methods, 2016, 13(7): 557-562. doi: 10.1038/nmeth.3891 pmid: 27240257
[4]	Hirano M, Ando R, Shimozono S, Sugiyama M, Takeda N, Kurokawa H, Deguchi R, Endo K, Haga K, Takai-Todaka R, Inaura S, Matsumura Y, Hama H, Okada Y, Fujiwara T, Morimoto T, Katayama K, Miyawaki A. A highly photostable and bright green fluorescent protein. Nat Biotechnol, 2022, 40(7): 1132-1142. doi: 10.1038/s41587-022-01278-2
[5]	Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, Wood MG, Learish R, Ohana RF, Urh M, Simpson D, Mendez J, Zimmerman K, Otto P, Vidugiris G, Zhu J, Darzins A, Klaubert DH, Bulleit RF, Wood KV. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol, 2008, 3(6): 373-382. doi: 10.1021/cb800025k pmid: 18533659
[6]	Hoelzel CA, Zhang X. Visualizing and manipulating biological processes by using HaloTag and SNAP-Tag technologies. Chembiochem, 2020, 21(14): 1935-1946. doi: 10.1002/cbic.202000037 pmid: 32180315
[7]	Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 2013, 8(11): 2281-2308. doi: 10.1038/nprot.2013.143 pmid: 24157548
[8]	Tamura R, Kamiyama D. CRISPR-Cas9-mediated knock- in approach to insert the GFP11 tag into the genome of a human cell line. Methods Mol Biol, 2023, 2564: 185-201. doi: 10.1007/978-1-0716-2667-2_8 pmid: 36107342
[9]	Surve S, Sorkin A. CRISPR/Cas9 gene editing of HeLa cells to tag proteins with mNeonGreen. Bio Protoc, 2022, 12(10): e4415. doi: 10.21769/BioProtoc.4415 pmid: 35813028
[10]	Koch B, Nijmeijer B, Kueblbeck M, Cai Y, Walther N, Ellenberg J. Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing. Nat Protoc, 2018, 13(6): 1465-1487. doi: 10.1038/nprot.2018.042 pmid: 29844520
[11]	Cabantous S, Terwilliger TC, Waldo GS. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol, 2005, 23(1): 102-107. doi: 10.1038/nbt1044 pmid: 15580262
[12]	Feng SY, Sekine S, Pessino V, Li H, Leonetti MD, Huang B. Improved split fluorescent proteins for endogenous protein labeling. Nat Commun, 2017, 8(1): 370. doi: 10.1038/s41467-017-00494-8 pmid: 28851864
[13]	Alford SC, Ding YD, Simmen T, Campbell RE. Dimerization-dependent green and yellow fluorescent proteins. ACS Synth Biol, 2012, 1(12): 569-575. doi: 10.1021/sb300050j pmid: 23656278
[14]	Rodriguez EA, Campbell RE, Lin JY, Lin MZ, Miyawaki A, Palmer AE, Shu XK, Zhang J, Tsien RY. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem Sci, 2017, 42(2): 111-129. doi: S0968-0004(16)30173-6 pmid: 27814948
[15]	Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 1996, 173(1 Spec No):33-38. doi: 10.1016/0378-1119(95)00685-0 pmid: 8707053
[16]	Zacharias DA, Violin JD, Newton AC, Tsien RY. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science, 2002, 296(5569): 913-916. doi: 10.1126/science.1068539 pmid: 11988576
[17]	Shaner NC, Lambert GG, Chammas A, Ni YH, Cranfill PJ, Baird MA, Sell BR, Allen JR, Day RN, Israelsson M, Davidson MW, Wang JW. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods, 2013, 10(5): 407-409. doi: 10.1038/nmeth.2413 pmid: 23524392
[18]	Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol, 2004, 22(12): 1567-1572. doi: 10.1038/nbt1037 pmid: 15558047
[19]	Merzlyak EM, Goedhart J, Shcherbo D, Bulina ME, Shcheglov AS, Fradkov AF, Gaintzeva A, Lukyanov KA, Lukyanov S, Gadella TWJ, Chudakov DM. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods, 2007, 4(7): 555-557. doi: 10.1038/nmeth1062 pmid: 17572680
[20]	Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M, Aumonier S, Gotthard G, Royant A, Hink MA, Gadella TWJ. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods, 2017, 14(1): 53-56. doi: 10.1038/nmeth.4074 pmid: 27869816
[21]	Mukherjee S, Hung ST, Douglas N, Manna P, Thomas C, Ekrem A, Palmer AE, Jimenez R. Engineering of a brighter variant of the fusionRed fluorescent protein using lifetime Flow Cytometry and structure-guided mutations. Biochemistry, 2020, 59(39): 3669-3682. doi: 10.1021/acs.biochem.0c00484
[22]	Leonetti MD, Sekine S, Kamiyama D, Weissman JS, Huang B. A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proc Natl Acad Sci USA, 2016, 113(25): E3501-E3508.
[23]	Los GV, Wood K. The HaloTag: a novel technology for cell imaging and protein analysis. Methods Mol Biol, 2007, 356: 195-208. pmid: 16988404
[24]	Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol, 2003, 21(1): 86-89. pmid: 12469133
[25]	Juillerat A, Gronemeyer T, Keppler A, Gendreizig S, Pick H, Vogel H, Johnsson K. Directed evolution of O6- alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo. Chem Biol, 2003, 10(4): 313-317. doi: 10.1016/S1074-5521(03)00068-1
[26]	Gautier A, Juillerat A, Heinis C, Corrêa IR, Kindermann M, Beaufils F, Johnsson K. An engineered protein tag for multiprotein labeling in living cells. Chem Biol, 2008, 15(2): 128-136. doi: 10.1016/j.chembiol.2008.01.007
[27]	Grimm JB, Brown TA, English BP, Lionnet T, Lavis LD. Synthesis of Janelia Fluor HaloTag and SNAP-Tag ligands and their use in cellular imaging experiments. Methods Mol Biol, 2017, 1663: 179-188. doi: 10.1007/978-1-4939-7265-4_15 pmid: 28924668
[28]	Trinh R, Gurbaxani B, Morrison SL, Seyfzadeh M. Optimization of codon pair use within the (GGGGS)3 linker sequence results in enhanced protein expression. Mol Immunol, 2004, 40(10): 717-722. pmid: 14644097
[29]	van Rosmalen M, Krom M, Merkx M. Tuning the flexibility of glycine-serine linkers to allow rational design of multidomain proteins. Biochemistry, 2017, 56(50): 6565-6574. doi: 10.1021/acs.biochem.7b00902 pmid: 29168376
[30]	Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9- crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA, 2012, 109(39): E2579-E2586.
[31]	Bukhari H, Müller T. Endogenous fluorescence tagging by CRISPR. Trends Cell Biol, 2019, 29(11): 912-928. doi: S0962-8924(19)30140-0 pmid: 31522960
[32]	Ratz M, Testa I, Hell SW, Jakobs S. CRISPR/Cas9- mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells. Sci Rep, 2015, 5: 9592. doi: 10.1038/srep09592
[33]	Hendel A, Kildebeck EJ, Fine EJ, Clark J, Punjya N, Sebastiano V, Bao G, Porteus MH. Quantifying genome- editing outcomes at endogenous loci with SMRT sequencing. Cell Rep, 2014, 7(1): 293-305. doi: 10.1016/j.celrep.2014.02.040 pmid: 24685129
[34]	Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, Olsen KM, Gregg A, Noggle S, Tessier-Lavigne M.Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature, 2016, 533(7601): 125-129. doi: 10.1038/nature17664
[35]	Concordet JP, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res, 2018, 46(W1): W242-W245. doi: 10.1093/nar/gky354
[36]	Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol, 2016, 34(2): 184-191. doi: 10.1038/nbt.3437 pmid: 26780180
[37]	He KM, Marsland R, Upadhyayula S, Song E, Dang S, Capraro BR, Wang WM, Skillern W, Gaudin R, Ma MH, Kirchhausen T. Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic. Nature, 2017, 552(7685): 410-414. doi: 10.1038/nature25146
[38]	He KM, Song E, Upadhyayula S, Dang S, Gaudin R, Skillern W, Bu K, Capraro BR, Rapoport I, Kusters I, Ma MH, Kirchhausen T. Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic. J Cell Biol, 2020, 219(3): e201908142. doi: 10.1083/jcb.201908142
[39]	Li GL, Yang SX, Wu ZF, Zhang XW. Recent developments in enhancing the efficiency of CRISPR/Cas9-mediated knock-in in animals. Hereditas(Beijing), 2020, 42(7): 641-656.
	李国玲, 杨善欣, 吴珍芳, 张献伟. 提高CRISPR/Cas9介导的动物基因组精确插入效率研究进展. 遗传, 2020, 42(7): 641-656.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

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

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

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 39

相关文章 14

编辑推荐

Metrics

[1]	严婷婷, 张蕾, 李余动, 梁新乐. 基于微信的“微生物遗传育种实验”混合式教学模式探究[J]. 遗传, 2018, 40(7): 601-606.
[2]	李智方, 冯冲, 纪慧丽, 石宁宁, 宋小凤, 赵勤丽, 龙川, 潘登科, 杨小淦. 绿色荧光蛋白在α-1,3半乳糖基转移酶敲除猪组织器官的表达分析[J]. 遗传, 2015, 37(12): 1211-1217.
[3]	张鹏，杨珍珍，窦红伟，李伟杭，律波，Bolund Lars，杜玉涛，谭萍萍，马润林. 利用改进的手工克隆技术生产转GFP基因猪克隆胚胎[J]. 遗传, 2011, 33(5): 527-532.
[4]	李姗姗，雍静茹，齐云玲，章颖，赵亮，夏士林，李东，王慧利，包其郁，李佩珍. 螺旋藻耐盐相关基因启动子区功能分析[J]. 遗传, 2011, 33(10): 1134-1140.
[5]	刘长青，郭俣，刘帅，包阿东，陆涛峰，刘洪坤，关伟军，马月辉 . 北京油鸡α1-AGP基因结构与表达特征研究[J]. 遗传, 2009, 31(6): 620-628.
[6]	徐纪明，向太和. 含gfp植物转基因表达载体的构建及在矮牵牛转基因不定根中的高效表达[J]. 遗传, 2008, 30(8): 1069-1074.
[7]	沈卫锋，翁宏飚，牛宝龙，何丽华，刘岩，齐晓朋，孟智启. 根癌农杆菌介导的灰葡萄孢菌遗传转化研究[J]. 遗传, 2008, 30(4): 515-520.
[8]	刘和，陈英旭，张文波，金勇丰. PCR一步法构建融合蛋白基因fpg[J]. 遗传, 2004, 26(4): 525-528.
[9]	许文明，张思仲，邱为民，何国平，刘运强，马用信，孙岩. ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位[J]. 遗传, 2004, 26(4): 451454-451454.
[10]	龙华，木下政人. GFP标记在转基因青鳉同系繁殖纯化中的应用[J]. 遗传, 2003, 25(4): 409-413.
[11]	李扬，吴凯峰，郭旭东，郭继彤，旭日干. 脂质体介导外源基因体外转染牛胎儿成纤维细胞条件的优化 [J]. 遗传, 2002, 24(6): 653-655.
[12]	黄国存，张寒霜，高鹏，李俊兰，朱生伟，孙敬三. GFP基因在棉花转化中的应用[J]. 遗传, 2001, 23(2): 131-200.
[13]	陈智毅，李清兵，陈列辉，廖森泰，李宝瑜，廖琼香. 利用CLSM检测GFP基因在家蚕的瞬时表达[J]. 遗传, 2000, 22(5): 303-304.
[14]	曾位森，夏宁邵，罗琛，谢卫兵，丁亮，黄宗平，曾定. 利用GFP示踪细胞内源性P53活性检测DNA损伤[J]. 遗传, 1999, 21(3): 5-710.