利用CRISPR/Cas9技术构建基因编辑大鼠模型

doi:10.16288/j.yczz.22-354

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Hereditas(Beijing) ›› 2023, Vol. 45 ›› Issue (1): 78-87.doi: 10.16288/j.yczz.22-354

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Generation of genetically modified rat models via the CRISPR/Cas9 technology

Meizhen Liu(), Liren Wang(), Yongmei Li, Xueyun Ma, Honghui Han, Dali Li()

School of Life Sciences, East China Normal University, Shanghai 200241, China

Received:2022-11-10 Revised:2022-12-23 Online:2023-01-20 Published:2023-01-01
Contact: Li Dali E-mail:mzliu@bio.ecnu.edu.cn;lrwang@bio.ecnu.edu.cn;dlli@bio.ecnu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China(32025023);Supported by the National Natural Science Foundation of China(81873685);Supported by the National Natural Science Foundation of China(31971366);the National Key Research and Development Program of China(2019YFA0110802);the National Key Research and Development Program of China(2019YFA0802802);the National Key Research and Development Program of China(2019YFA0802802);the Shanghai Municipal Commission for Science and Technology(20140900200);the Animal Center of East China Normal University(011)

Abstract

Abstract:

The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.

Key words: rat, CRISPR/Cas9, genome editing, gene knockout

Meizhen Liu, Liren Wang, Yongmei Li, Xueyun Ma, Honghui Han, Dali Li. Generation of genetically modified rat models via the CRISPR/Cas9 technology[J]. Hereditas(Beijing), 2023, 45(1): 78-87.

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References 10

[1]	Geurts AM, Moreno C. Zinc-finger nucleases: new strategies to target the rat genome. Clin Sci Lond Engl, 2010, 119(8): 303-311.
[2]	Kottaisamy CPD, Raj DS, Prasanth Kumar V, Sankaran U. Experimental animal models for diabetes and its related complications-a review. Lab Anim Res, 2021, 37(1): 23.
[3]	Qiu Z, Liu M, Chen Z, Shao Y, Pan H, Wei G, Yu C, Zhang L, Li X, Wang P, Fan HY, Du B, Liu B, Liu M, Li D. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases. Nucleic Acids Res, 2013, 41(11): e120-e120.
[4]	Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, Zhao Y, Liu M. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013, 31(8): 681-683.
[5]	Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012, 337(6096): 816-821.
[6]	Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823.
[7]	Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 2013, 153(4): 910-918.
[8]	Zheng R, Li Y, Wang L, Fang X, Zhang J, He L, Yang L, Li D, Geng H.CRISPR/Cas9-mediated metabolic pathway reprogramming in a novel humanized rat model ameliorates primary hyperoxaluria type 1. Kidney Int, 2020, 98(4): 947-957.
[9]	Shao Y, Guan Y, Wang L, Qiu Z, Liu M, Chen Y, Wu L, Li Y, Ma X, Liu M, Li D. CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nat Protoc, 2014, 9(10): 2493-2512.
[10]	Guan Y, Ma Y, Li Q, Sun Z, Ma L, Wu L, Wang L, Zeng L, Shao Y, Chen Y, Ma N, Lu W, Hu K, Han H, Yu Y, Huang Y, Liu M, Li D. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med, 2016, 8(5): 477-488.

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实验材料名称	品牌	货号
Cas9 mRNA	Thermo Fisher	A29378
sgRNA	Genescript	EasyEdit sgRNA
20× RNAase Free TE buffer	Thermo Fisher	T11493
SD大鼠	上海斯莱克实验动物有限公司	NA
质粒PX260	Addgene	42229
KOD-plus-Neo DNA聚合酶	Toyobo	KOD-401
溴化乙锭	天根	RT203
sp6体外转录试剂盒	Life Technologies	AM1340
无核酸酶水	Promega	P1195
促性腺激素(PMSG)	Millipore	367222
绒毛促性腺激素(HCG)	Millipore	230734
M2溶液	Millipore	MR-015-D
透明质酸酶	Sigma	H4272
KSOM溶液	Millipore	MR-020P-5F
胚胎移植用矿物油	Sigam	M5310
异氟烷	瑞沃德	R510-22-10
Trizma盐酸盐胚胎注射等级	Sigma	T2663
EDTA胚胎注射等级	Sigma	E7889
水胚胎注射等级	Sigma	W3500
DPBS	Gibco	1490144
M16 medium	Millpore	MR-016-D
Tris base	Amresco	0497
蛋白酶K	Sigma	70663-5
乙酸钠	Sigma	539480
35 mm培养皿	Corning	430165
玻璃管	Sutter	B100-75-10
微毛细管	Sigma	P1049
微量上样针	Eppendorf	5242956003

名称	成分
蛋白酶K母液	20 mg/mL蛋白酶K水溶液
消化液	0.1 mol/L Tris base、0.05 mol/L EDTA、0.2 mol/L NaCl、0.2% (wt/vol) SDS
透明质酸酶M2 溶液	M2溶液中加入透明质酸母液，终浓度为0.06 μg/μL

名称	Heat*	Pull*	Vel*	Time*
固定吸管	817	0	20	200
纤维注射针	815	80	60	200

成分	体积(μL)	终浓度或总量
基因组DNA	1	100 ng
FastPfu fly缓冲液(5×)	10	1×
FastPfu fly	1
正向引物(10 μmol/L)	1	0.2 μmol/L
反向引物(10 μmol/L)	1	0.2 μmol/L
dNTP(10 mmol/L)	1	0.2 mmol/L
无核酸酶水	34

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Generation of genetically modified rat models via the CRISPR/Cas9 technology

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