CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane

Author:

Ondra Martin12ORCID,Lenart Lukas1,Centorame Amanda34,Dumut Daciana C34,He Alexander5,Zaidi Syeda Sadaf Zehra5,Hanrahan John W45ORCID,De Sanctis Juan Bautista1ORCID,Radzioch Danuta134ORCID,Hajduch Marian126ORCID

Affiliation:

1. Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University

2. Czech Advanced Technology and Research Institute, Palacky University

3. Faculty of Medicine and Health Sciences, McGill University

4. RI-MUHC, Montreal, Canada

5. Physiology, McGill University

6. Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, University Hospital Olomouc, Olomouc, Czech Republic

Abstract

CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.

Funder

The Quebec Ministry of Economy and Innovation

Ministry of School and Education of the Czech Republic

CIHR

RI-MUHC

Publisher

Life Science Alliance, LLC

Subject

Health, Toxicology and Mutagenesis,Plant Science,Biochemistry, Genetics and Molecular Biology (miscellaneous),Ecology

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3