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Electroporation conditions for CRISPR/Cas9-mediated genome editing
- NUCLEAR TECHNIQUES Vol. 41, Issue 8, Article number: 080502(2018)
- 作者机构：
1.中国科学院上海应用物理研究所 物理生物学研究室 中国科学院微观界面物理与探测重点实验室 上海同步辐射光源生物成像中心 嘉定园区 上海 201800
2.中国科学院上海应用物理研究所 熔盐化学与工程技术部 嘉定园区 上海 201800
3.中国科学院大学 北京 100049
- 作者简介：
- 基金信息：National Key Research and Development Program;the Open Large Infrastructure Research of Chinese Academy of Sciences;National Natural Science Foundation of China;National Natural Science Foundation of China;Instrument Developing Project of Chinese Academy of Sciences;the Youth Innovation Promotion Association of Chinese Academy of Sciences;the China Postdoctoral Science Foundation;the China Postdoctoral Science Foundation
DOI：10.11889/j.0253-3219.2018.hjs.41.080502
CLC： TL99Received：28 March 2018，
Revised：09 April 2018，
Published：2018-08
- 稿件说明：
移动端阅览
Kai XIA, Ling HAN, Huating KONG, et al. Electroporation conditions for CRISPR/Cas9-mediated genome editing[J]. Nuclear techniques, 2018, 41(8): 080502
Kai XIA, Ling HAN, Huating KONG, et al. Electroporation conditions for CRISPR/Cas9-mediated genome editing[J]. Nuclear techniques, 2018, 41(8): 080502 DOI： 10.11889/j.0253-3219.2018.hjs.41.080502.
摘要
直接向细胞内输运CRISPR/Cas9核糖核蛋白复合物（Cas9 RNP）对于进行各种基因编辑、基因表达调控和DNA/RNA成像具有重要意义。电转是向细胞内导入Cas9 RNP的重要方法。本工作研究了电压、脉冲长度及脉冲次数等电转条件对Cas9 RNP细胞内基因编辑效率的影响，评估了不同电转条件下的细胞存活率。结果显示：电压为1300 V，脉冲次数为1，脉冲宽度为30 ms时，Cas9 RNP具有较高的基因编辑效率且生物安全性较好。本工作为CRISPR/Cas9复合物进一步的生物医学应用提供了指导依据。
Abstract
Background
2
Direct cellular delivery of CRISPR/Cas9 complexes is of great significance for genome editing
gene expression regulation
and DNA/RNA imaging. Electroporation is an important approach for the delivery of Cas9 ribonucleoproteins (RNP).
Purpose
2
This study aims to screen the optimal electroporation condition for CRISPR/Cas9-mediated genome editing.
Methods
2
SDS PAGE
Western bolt
Agarose gel electrophoresis and T7E1 cleavage assay were used to investigate the CRISPR/Cas9-mediated gene editing efficiency under various electroporation conditions. 3-(4
5-dimethyl-2-thiazolyl)-2
5-diphenyl-2-H-tetrazolium bromide (MTT) and Caspase-3 activity determination methods were used to investigate the biocompatibility of various electroporation conditions.
Results
2
When the voltage is 1 300 V
pulse number is 1
and pulse width is 30 ms
we get the optimal genome editing efficiency with low cellular toxicity.
Conclusion
2
Our work provides useful information of using CRISPR/Cas9 complexes for further biomedical applications.
关键词
Keywords
references
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