电转条件对Cas9核糖核蛋白基因编辑效率的影响

夏凯; 韩玲; 孔华庭; 闫庆龙; 张瑜; 诸颖

doi:10.11889/j.0253-3219.2018.hjs.41.080502

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电转条件对Cas9核糖核蛋白基因编辑效率的影响

核物理、交叉学科研究 | 更新时间：2021-05-19

- 电转条件对Cas9核糖核蛋白基因编辑效率的影响
- Electroporation conditions for CRISPR/Cas9-mediated genome editing
- 核技术 2018年41卷第8期文章编号：080502
- 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：
  
  [ "夏凯, 女, 1989年出生, 2015年于中国科学院上海应用物理研究所获硕士学位, 研究领域为纳米生物效应", "XIA Kai, female, born in 1989, graduated from Shanghai Institute of Applied Physics, Chinese Academy of Sciences with a master's degree in 2015, focusing on bio-effects of nanomaterials" ]
  诸颖, E-mail:zhuying@sinap.ac.cn ZHU Ying, E-mail:zhuying@sinap.ac.cn
- 基金信息：
  
  国家重点研发计划(2016YFA0400902);国家自然科学基金(11675251);国家自然科学基金(21390414);中国科学院青年创新促进会(2016236);中国博士后科学基金(2016M601679);中国博士后科学基金(2018M632189)
- DOI：10.11889/j.0253-3219.2018.hjs.41.080502
  中图分类号： TL99
- 收稿日期：2018-03-28，
  
  修回日期：2018-04-09，
  
  纸质出版日期：2018-08
- 稿件说明：
移动端阅览
夏凯, 韩玲, 孔华庭, 等. 电转条件对Cas9核糖核蛋白基因编辑效率的影响[J]. 核技术, 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
夏凯, 韩玲, 孔华庭, 等. 电转条件对Cas9核糖核蛋白基因编辑效率的影响[J]. 核技术, 2018,41(8):080502 DOI： 10.11889/j.0253-3219.2018.hjs.41.080502.

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

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

This study aims to screen the optimal electroporation condition for CRISPR/Cas9-mediated genome editing.

Methods

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

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

Our work provides useful information of using CRISPR/Cas9 complexes for further biomedical applications.

关键词

Keywords

references

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