CRISPR/Cas9‐Mediated Genome Editing in Epstein‐Barr Virus‐Transformed Lymphoblastoid B‐Cell Lines

Sizun Jiang1, Liang Wei Wang2, Michael J. Walsh2, Stephen J. Trudeau2, Catherine Gerdt2, Bo Zhao2, Benjamin E. Gewurz2

1 Present Address: Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine, Stanford, 2 Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 31.12
DOI:  10.1002/cpmb.51
Online Posting Date:  January, 2018
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Epstein‐Barr virus (EBV) efficiently transforms primary human B cells into immortalized lymphoblastoid cell lines (LCLs), which are extensively used in human genetic, immunological and virological studies. LCLs provide unlimited sources of DNA for genetic investigation, but can be difficult to manipulate, for instance because low retroviral or lentiviral transduction frequencies hinder experiments that require co‐expression of multiple components. This unit details Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 engineering for robust LCL genome editing. We describe the generation and delivery of single‐guide RNAs (sgRNAs), or dual‐targeting sgRNAs, via lentiviral transduction of LCLs that stably express Cas9 protein. CRISPR/Cas9 editing allows LCL loss‐of‐function studies, including knock‐out of protein‐coding genes or deletion of DNA regulatory elements, and can be adapted for large‐scale screening approaches. Low transfection efficiencies are a second barrier to performing CRISPR editing in LCLs, which are not typically lipid‐transfectable. To circumvent this barrier, we provide an optimized protocol for LCL nucleofection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) as an alternative route to achieve genome editing in LCLs. These editing approaches can also be employed in other B‐cell lines, including Burkitt lymphoma and diffuse large B‐cell lymphoma cells, and are highly reproducible. © 2018 by John Wiley & Sons, Inc.

Keywords: Burkitt lymphoma; CRISPR; Epstein‐Barr virus; enhancer; genome editing; lymphoblastoid

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Table of Contents

  • Introduction
  • Basic Protocol 1: Establishment and Validation of Cas9‐Expressing Stable LCLs
  • Support Protocol 1: Lentivirus Packaging and Infection of LCLs
  • Basic Protocol 2: Design and Cloning of Single Target sgRNAs
  • Basic Protocol 3: Design and Cloning of Dual Targeting sgRNAS into the Same Vector
  • Alternate Protocol 1: Nucleofection of Ribonucleoprotein Complexes into LCLs for Genome Editing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Establishment and Validation of Cas9‐Expressing Stable LCLs

  • Cultured lymphoblastoid cells
  • R10 medium (see recipe)
  • Aliquots of prepared lentiviruses from lentiCas9‐Blast (Addgene, cat. no. 52962) and pXPR_011 (Addgene, cat. no. 59702): for details on how to package lentiviruses from these plasmids, please refer to protocol 2
  • 10% bleach in H 2O
  • R10‐Blasticidin (see recipe)
  • R10‐Puromycin (see recipe)
  • Phosphate‐buffered saline (PBS; ThermoFisher Scientific, cat. no. 14190136)
  • BSL2/BSL2+ certified tissue culture hood
  • 37°C tissue culture incubator supplemented with 5% CO 2
  • 15‐ml conical tubes
  • Centrifuge suitable for 15‐ml conical tubes
  • 37°C water bath
  • Clear 6‐well plates, sterile (Sigma‐Aldrich, cat. no. CLS3516‐10EA)
  • Fluorescence microscope equipped with a green emission filter (typically in the 500 nm‐550 nm range)
  • 1.5‐ml microcentrifuge tubes
  • Flow cytometer
  • 0.45‐μm filter
  • Multichannel pipette
  • Clear 96‐well plates, sterile (Sigma‐Aldrich, cat. no. CLS3300‐50EA)
OPTIONAL: Cas9 mouse monoclonal antibody clone 7A9‐3A3 (Active Motif, cat. no. 61578)CAUTION: Ensure that cells are free of mycoplasma by testing regularly and practicing good aseptic techniques. The presence of mycoplasma can severely skew experimental outcomes.CAUTION: Although lentiviruses used in this protocol are replication‐defective, handle with care. Always bleach anything that comes in contact with lentivirus stocks before disposal (e.g., pipette tips).NOTE: Warm all media to 37°C before addition to LCLs.

Support Protocol 1: Lentivirus Packaging and Infection of LCLs

  • 293FT cells (Thermo Fisher Scientific, cat. no. R70007) or HEK‐293T cells (ATCC CRL‐3216)
  • DMEM medium
  • pCMV‐VSV‐G (Addgene, cat. no.8454); dilute plasmid to 30 ng/µl
  • psPAX2 (Addgene, cat. no. 12260); dilute plasmid to 80 ng/μl
  • lentiCas9‐Blast (Addgene, cat. no. 52962); dilute plasmid to 100 ng/μl
  • pXPR_011 (Addgene, cat. no. 59702); dilute plasmid to 100 ng/μl
  • OPTI‐MEM medium (Thermo Fisher, cat. no. 31985062)
  • TransIT‐LT1 Transfection Reagent (Mirus, cat. no. MIR 2306)
  • R10 medium (see recipe)
  • 10% bleach in H 2O
  • BSL2/BSL2+ certified tissue culture hood
  • Clear 6‐well plates, sterile (Sigma‐Aldrich, cat. no. CLS3516‐10EA)
  • 37°C tissue culture incubator supplied with 5% CO 2
  • Sterile 1.5‐ml microcentrifuge tubes
  • Confocal microscope
  • 0.45‐μm filter
CAUTION: All steps can be performed in sterile cell culture conditions.OPTIONAL: Plasmid DNA can be purified using an endotoxin‐free plasmid prep kit, to ensure transfection‐grade DNA plasmid. While essential for B‐cell transfection, it is optional for HEK‐293 cell transfection.CAUTION: Although lentiviruses used in this protocol are replication‐defective, handle with care. Always use 10% bleach to disinfect anything that comes in contact with lentivirus stocks before disposal (e.g., pipette tips).CAUTION: Keep TransIT‐LT1 at 4°C until required.NOTE: All dilutions can be performed with molecular‐grade water or TE buffer.NOTE: Pre‐warm all media to 37°C before the experiment.NOTE: Aliquot OPTI‐MEM media into 15 ml tubes to avoid cross‐contamination.NOTE: For large numbers of plasmids, perform plasmid‐transfection reagent mixtures in a sterile 96 well plate for easy tracking and access.NOTE: Always include a GFP control (e.g., pXPR_011) to check for transfection and transduction efficiency.

Basic Protocol 2: Design and Cloning of Single Target sgRNAs

  • lentiGuide‐Puro (Addgene, cat. no. 52963); dilute plasmid to 1000 ng/μl
  • FastDigest Esp3I (BsmBI) (Thermo Fisher Scientific, cat. no. FD0454)
  • 10× Green FastDigest Buffer (included in Fast Digest Esp3I kit)
  • FastAP (Thermo Fisher Scientific, cat. no. FD0454)
  • 100 mM DTT (prepare fresh as 1 M stocks and store aliquots at −20°C)
  • H 2O (Molecular Biology Grade)
  • Elution buffer (included in NucleoSpin Gel and PCR Clean‐up kit)
  • NucleoSpin® Gel and PCR Clean‐up kit (Macherey‐Nagel, cat. no. 740609.50)
  • sgRNA oligonucleotide pairs (100 μM)
  • T4 ligase (NEB, cat. no. M0202S)
  • T4 PNK (NEB, cat. no. M0201S)
  • One Shot Stbl3 Chemically Competent E.coli (ThermoFisher Scientific, cat. no. C737303)
  • LB/ampicillin plates
  • 37°C incubator
  • Thermal cycler
  • PCR thermal cycler strip tubes
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5, Voytas, )

Basic Protocol 3: Design and Cloning of Dual Targeting sgRNAS into the Same Vector

  • Digested lentiGuide‐Puro, diluted to 50 ng/μl (from protocol 3)
  • PrimeSTAR® HS DNA Polymerase premix (Takara, cat. no. R040A)
  • Bridge dsDNA block for H1/sU6 promoter, diluted to 0.01 ng/μl; we recommend ordering these as gBlocks from IDT, and storing as a 1 ng/μl stock at ‐20C (see Supplementary Data for sequence)
  • Molecular biology grade water
  • Agarose gel (1% in TAE)
  • NucleoSpin® Gel and PCR Clean‐up (Macherey‐Nagel, cat, no, 740609.50)
  • In‐Fusion® HD Cloning Plus (Takara, cat. no. 638909)
  • Ice
  • One Shot Stbl3 Chemically Competent E.coli (ThermoFisher Scientific, cat. no. C737303) containing:SOC medium
  • LB/ampicillin plates
  • Qiaprep Spin Miniprep kit (Qiagen, cat. no. 27104)
  • Thermal cycler
Design and cloning of dual sgRNAs

Support Protocol 2:

  • QIAamp DNA Mini Kit (Qiagen, cat. no. 51304) containing:
  • ‐Buffers ATL, AL, AW1, AW2, AE, and Proteinase K
  • QIAamp elution columns
  • 2‐ml collection tubes
  • 100% Ethanol (Molecular Biology Grade)
  • PrimeSTAR® HS DNA Polymerase premix (Takara, cat. no. R040A)
  • PCR amplification primers flanking deletion region, diluted to 10 μM
  • Water (Molecular Biology Grade)
  • 1% agarose gel
  • 1.5‐ml microcentrifuge tubes
  • Thermal cycler
NOTE: Primers should be designed with a tool such as IDT's PrimerQuest (see Internet Resources)

Alternate Protocol 1: Nucleofection of Ribonucleoprotein Complexes into LCLs for Genome Editing

  • CRISPRevolution sgRNA EZ Kit with Cas9 2NLS Nuclease Add‐On (Synthego) containing:
  • Cas9 2NLS Nuclease (300 pmol)
  • sgRNA (3nmol)
  • Tris‐EDTA buffer
  • Nuclease‐free H 2O
  • Ice
  • SF Cell Line 4D‐Nucleofector® X Kit S (32 RCT) (Lonza, cat. no. V4XC‐2032) containing:
  • SF Nucleofector® solution with added supplement
  • 4D‐Nucleofector® X Kit S strip
  • R10 medium (see recipe)
  • BSL2/BSL2+ certified Tissue Culture hood
  • 15‐ml conical tubes
  • Centrifuge
  • Pipettes
  • 4D‐Nucleofector™ Core Unit (Lonza, cat. no. AAF‐1002B)
  • 4D‐Nucleofector™ System X Unit (Lonza, cat. no. AAF‐1002X)
  • Clear 24‐well plates, sterile (Sigma‐Aldrich, cat. no. CLS3524‐100EA)
  • 37°C tissue culture incubator supplemented with 5% CO 2
  • Fluorescent light microscope
  • 1.5‐ml RNase‐free microcentrifuge tubes
CAUTION: All steps should be done in a cell culture hood with the practice of aseptic techniques.
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Internet Resources
  The Benchling online Web tool: This free online Web tool provides an easy interface for users to input a sequence of DNA, and rapidly returns potential sgRNA targets with on and off‐target scores.‐attachment_IPB7ZL_hJcbm.pdf.
  The Zhang Lab CRISPR cloning protocol: This is a detailed protocol from the Zhang laboratory on cloning an sgRNA into the lentiCRISPR or lentiGuide‐Puro vectors.‐tools/sgrna‐design.
  Broad Institute sgRNA design tool: This site picks and ranks sgRNAs against provided target sequences, and designs sgRNAs for use with either S. pyogenes or S. aureus Cas9.
  IDT PrimerQuest primer design tool: This tool is useful for optimal design and ordering of PCR primers.
  The TIDE (Tracking of Indels by Decomposition) tool: TIDE allows for easy quantification of double strand breaks from sequencing of PCR products from a heterogeneous pool.
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