Modulating Gene Expression in Epstein‐Barr Virus (EBV)‐Positive B Cell Lines with CRISPRa and CRISPRi

Liang Wei Wang1, Stephen J. Trudeau1, Chong Wang2, Catherine Gerdt2, Sizun Jiang3, Bo Zhao2, Benjamin E. Gewurz1

1 Program in Virology, Harvard Medical School, Harvard University, Boston, Massachusetts, 2 Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, 3 Current affiliation: Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 31.13
DOI:  10.1002/cpmb.50
Online Posting Date:  January, 2018
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Epstein‐Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease‐deficient Cas9 (dCas9)‐VP64 (Cas9 associated with CRISPRa) or dCas9‐KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus. The ribonucleoprotein complex formed by the dCas9 molecule and its cognate sgRNA enables sequence‐specific binding at a promoter or enhancer of interest to affect the expression of genes regulated by the targeted promoter or enhancer. © 2018 by John Wiley & Sons, Inc.

Keywords: CRISPR; Epstein‐Barr virus; enhancer; promoter; transcription regulation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation and Validation of CRISPRa‐ and CRISPRi‐Competent B Cell Lines
  • Basic Protocol 2: Designing and Cloning of Single‐Guide RNAs for CRISPRa and CRISPRi
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Generation and Validation of CRISPRa‐ and CRISPRi‐Competent B Cell Lines

  • GM12878 lymphoblastoid B cell line (LCL; ENCODE Tier 1 EBV‐positive lymphoblastoid B cells obtained from Coriell, GM12878; maintain GM12878 LCLs at 1 × 105 cells/ml R10 medium at 37°C in a humidified incubator supplemented with 5% CO 2; passage cells every 2 to 3 days to maintain ideal cell density and culture medium pH)
  • R10 medium (see recipe)
  • R10‐blasticidin (see recipe)
  • 1× Laemmli sample buffer (see recipe)
  • R10‐puromycin (see recipe)
  • PBS (Gibco)
  • Freezing medium (see recipe)
  • Lentiviral supernatants (pXPR_109 for dCas9‐VP64; pXPR_121 for KRAB‐dCas9; see Jiang, Wang, et al., , unit 31.12, Support Protocol 1 for details on how to package lentiviruses using these plasmids)
  • Polybrene reagent (optional; EMD Millipore, cat. no. TR‐1003‐G)
  • Alexa Fluor 488 anti‐human CD19 antibody (eBioscience, cat. no. 53‐0199‐41)
  • FITC anti‐human CD86 monoclonal antibody (BD Biosciences, cat. no. 557343)
  • PE anti‐human CD95 (FAS) antibody (Biolegend, cat. no. 305607)
  • Mouse anti‐Cas9 monoclonal antibody, optional (clone 7A9‐3A3, Active Motif, cat. no. 61578)
  • 10% bleach in water (H 2O)
  • T25 and T75 cell culture flasks
  • Clear 6‐well plates, sterile (Sigma‐Aldrich, cat. no. CLS3516‐10EA)
  • Clear 96‐well plates, sterile (Sigma‐Aldrich, cat. no. CLS3300‐50EA)
  • 15‐ml Falcon tubes, sterile
  • 1.5‐ml microcentrifuge tubes, sterile
  • Centrifuge (suitable for 15‐ml Falcon tubes)
  • 37°C tissue culture incubator supplemented with 5% CO 2
  • BSL2/BSL2+ certified tissue culture hood
  • Flow cytometer
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, , unit 10.2) and immunoblotting (Ni, Xu, and Gallagher, , unit 10.8)
CAUTION: Ensure that cells are mycoplasma‐free by testing regularly and practicing good aseptic techniques.CAUTION: Although lentiviruses used in this protocol are replication‐defective, handle with care. Always bleach all pipets, tips, and related equipment, which have come into contact with lentiviral supernatants before disposal.NOTE: Pre‐warm medium at 37°C before addition to LCLs.

Basic Protocol 2: Designing and Cloning of Single‐Guide RNAs for CRISPRa and CRISPRi

  • Oligonucleotides (see sections A and B for procedures to design sgRNAs against promoters and enhancers, respectively), purified by standard desalting and diluted to 100 μM with nuclease‐free water
  • FastDigest BsmBI or Esp3I (Fermentas)
  • 10× FastDigest buffer (Fermentas)
  • pXPR_501 or pLentiGuide‐Puro
  • Double‐distilled water (ddH 2O)
  • QIAquick Gel Extraction kit or Macherey‐Nagel Nucleospin Gel and PCR Clean‐up kit
  • T4 DNA ligase (New England BioLabs)
  • 10× T4 ligase buffer (New England BioLabs)
  • One Shot Stbl3 chemically competent E. coli (Invitrogen)
  • Sterile S.O.C. medium (Thermo Fisher Scientific, Invitrogen brand)
  • QIAprep Spin Miniprep kit (Qiagen)
  • LB‐ampicillin (100 μg/ml) plates
  • NanoDrop spectrophotometer
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Internet Resources‐tools/sgrna‐design‐crisprai
  The Broad Institute's Genetic Perturbation Platform CRISPRa and CRISPRi sgRNA design tool. This free online tool allows users to design CRISPRa and CRISPRi sgRNAs for use with either SpCas9 or Staphylococcus aureus Cas9 (SaCas9) in human and murine cells, and also returns on‐ and off‐target information for the candidate sgRNAs.
  WashU Epigenome Browser LCL ChIP‐seq and ChIA‐PET Tracks. This session enables visualization of epigenomic data (Zhou et al., ) used in Jiang, Zhou, et al. ().
  Benchling: This free online portal enables the user to put in defined coordinates of the human genome and obtain a list of candidate sgRNAs with associated on‐ and off‐target scores (Doench et al., ; Hsu et al., ).‐attachment_YrytFfyNLHp5.pdf
  Zhang Lab's sgRNA cloning protocol: This document outlines the steps needed to generate sgRNA constructs.
  qPrimerDepot: This database rapidly generates qPCR primer sequences that can be used for validation of CRISPRa‐ and CRISPRi‐targeted transcripts.
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