Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

CRISPR-dCas9-KRAB-mediated suppression of super-enhancer

SX Sinan Xiong
JZ Jianbiao Zhou
TT Tze King Tan
TC Tae-Hoon Chung
TT Tuan Zea Tan
ST Sabrina Hui-Min Toh
NT Nicole Xin Ning Tang
YJ Yunlu Jia
YS Yi Xiang See
MF Melissa Jane Fullwood
TS Takaomi Sanda
WC Wee-Joo Chng
request Request a Protocol
ask Ask a question
Favorite

The dCas9-KRAB-T2A-mCherry plasmid was constructed by replacing the GFP expression cassette of dCas9-KRAB-T2A-GFP lentiviral vector (Addgene plasmid #71237) with mCherry sequence. The single guide RNAs (sgRNAs) targeting the PPP1R15B promoter or enhancer loci were designed using the online design tool Synthego (https://www.synthego.com/) and subsequently cloned into the BsmBI site of doxycycline-inducible gRNA vector FgH1tUTG (Addgene plasmid # 70183). The sgRNA sequences including non-targeting control are listed in Supplementary Data 4. Myeloma cells were infected with dCas9-KRAB-T2A-mCherry lentivirus and the mCherry positive cells were sorted using FACSAria Flow Cytometer (BD Biosciences). The cells that stably express dCas9-KRAB were next transduced with sgRNA-expressing constructs and were FACS sorted for double-positive cells (GFP + ;mCherry + ). Doxycycline (Dox, 1 µg/ml) was added following infection and the gene silencing effect was assessed by PCR and western blot.

Copyright and License information: The Author(s) ©2024
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A