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CRISPRi validation

QZ Quanyi Zhao
MD Michael Dacre
TN Trieu Nguyen
MP Milos Pjanic
BL Boxiang Liu
DI Dharini Iyer
PC Paul Cheng
RW Robert Wirka
JK Juyong Brian Kim
HF Hunter B. Fraser
TQ Thomas Quertermous
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We used dCas9 fused to KRAB to knockdown the enhancer region where candidate SNPs were located. The gRNAs targeting SNPs within the maximum 100 bp distance were designed using Benching online tools. Synthesized oligos were then cloned into pLV lentivirus vector containing dCas9-KRAB. For virus production, 8.5 × 105 HEK293T cells were plated in 6-well plate per well. The following day, plasmid encoding lentivirus was co-transfected with pMD2.G and pCMV-dR8.91 into the cells using Lipofectamine 3000 (Thermo Fisher, L3000015) according to the manufacturer’s instructions. ViralBoost Reagent (AlStem Cell Advancements, VB100) was added (1:500) when fresh media were added. Supernatant containing viral particles was collected 72 h after transfection and filtered. To knockdown, we plated 5 × 104 HACSMC cells in 6-well plate per well and cultured for 14 h and then added the virus supernatant to HCASMC for 12 h with 8 μg/mL polybrene. The cells were cultured for an additional 48 h for further experiments with medium change.

Copyright and License information: The Author(s) ©2020
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, 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 changes were made. 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/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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