Imperial College London
Alternate

Professor Mark Isalan - Deputy Head of Department

Faculty of Natural Sciences, Department of Life Sciences

Professor of Synthetic Biology
 
 
 
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Contact

 

+44 (0)20 7594 6482m.isalan

 
 
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Location

 

509Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Citation

BibTex format

@article{Grob:2018:10.1007/978-1-4939-7768-0_16,
author = {Grob, A and Marbiah, MM and Isalan, M},
doi = {10.1007/978-1-4939-7768-0_16},
journal = {Methods in Molecular Biology},
pages = {285--301},
title = {Functional insulator scanning of CpG islands to identify regulatory regions of promoters using CRISPR},
url = {http://dx.doi.org/10.1007/978-1-4939-7768-0_16},
volume = {1766},
year = {2018}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - The ability to mutate a promoter in situ is potentially a very useful approach for gaining insights into endogenous gene regulation mechanisms. The advent of CRISPR/Cas systems has provided simple, efficient, and targeted genetic manipulation in eukaryotes, which can be applied to studying genome structure and function.The basic CRISPR toolkit comprises an endonuclease, Cas9, and a short DNA-targeting sequence, made up of a single guide RNA (sgRNA). The catalytic domains of Cas9 are rendered active upon dimerization of Cas9 with sgRNA, resulting in targeted double stranded DNA breaks. Among other applications, this method of DNA cleavage can be coupled to endogenous homology-directed repair (HDR) mechanisms for the generation of site-specific editing or knockin mutations, at both promoter regulatory and gene coding sequences.A well-characterized regulatory feature of promoter regions is the high abundance of CpGs. These CpG islands tend to be unmethylated, ensuring a euchromatic environment that promotes gene transcription. Here, we demonstrate CRISPR-mediated editing of two CpG islands located within the promoter region of the MDR1 gene (Multi Drug Resistance 1). Cas9 is used to generate double stranded breaks across multiple target sites, which are then repaired while inserting the beta globin (β-globin) insulator, 5′HS5. Thus, we are screening through promoter regulatory sequences with a chromatin barrier element to identify functional regions via “insulator scanning.” Transcriptional and functional assessment of MDR1 expression provides evidence of genome engineering. Overall, this method allows the scanning of CpG islands to identify their promoter functions.
AU  - Grob,A
AU  - Marbiah,MM
AU  - Isalan,M
DO  - 10.1007/978-1-4939-7768-0_16
EP  - 301
PY  - 2018///
SN  - 1940-6029
SP  - 285
TI  - Functional insulator scanning of CpG islands to identify regulatory regions of promoters using CRISPR
T2  - Methods in Molecular Biology
UR  - http://dx.doi.org/10.1007/978-1-4939-7768-0_16
UR  - http://hdl.handle.net/10044/1/56918
VL  - 1766
ER  -
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