Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Summary

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

Abstract

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function—to produce large amounts of antibodies—can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Introduction

B cells are a subgroup of the lymphocyte lineage derived from hematopoietic stem cells. They perform a critical role in the adaptive humoral immune system by producing large amounts of antibodies in response to immune challenges¹. B cells are also precursors of memory B cells and the terminally differentiated, long-lived plasma cells, thereby providing lasting humoral immunity². Plasma cells, in particular, are unique among immune cells in their ability to produce large amounts of a specific antibody while surviving for years or decades³. Additionally, the ease of isolation from peripheral blood makes the B-cell lineage an excellent candidate for novel cell-based therapies⁴.

Previously, random integration methods, such as those using lentiviral vectors or a Sleeping Beauty transposon, have been used to engineer B cells for transgene delivery and expression^5,6,7,8. However, the non-specific nature of these approaches makes it difficult to study the biological functions of a specific gene in the B cells and carries an inherent risk of insertional mutagenesis and variable transgene expression and/or silencing in the therapeutic setting.

The CRISPR/Cas9 system is a powerful genome engineering tool that allows researchers to precisely edit the genome of various cells in numerous species. Recently, two groups, including our own, have successfully developed methods for ex vivo expansion and targeted genome engineering of primary human B cells^9,10. We will describe the process of purifying primary human B cells from a leukaphoresis sample. After that, we will describe our updated protocol for B-cell expansion and activation of isolated B cells. We will then describe a process for knocking out cluster of differentiation 19 (CD19), a specific B-cell receptor and a hallmark of B cells, by electroporation to introduce CRISPR/Cas9 mRNA together with CD19 sgRNA into activated B cells.

Cas9 mRNA gets translated and binds to the CD19 sgRNA to form a CRISPR/Cas9-sgRNA ribonucleoprotein complex (RNP). Subsequently, sgRNA in the complex leads Cas9 to create double-strand break (DSB) at the target sequence on exon 2 of the gene. The cells will repair the DSB by “non-homologous end joining” by introducing or deleting nucleotides, leading to frameshift mutation and causing the gene to be knocked out. We will then measure the loss of CD19 by flow cytometry and analyze indel formation by tracking of indels by decomposition (TIDE) analysis.

We will then describe the process of using CRISPR/Cas9 together with a recombinant AAV6 vector (rAAV6, a donor template for homology-directed repair (HDR)) to mediate site-specific insertion of enhanced green fluorescent protein (EGFP) at the adeno-associated virus integration site 1 (AAVS1) gene. The AAVS1 gene is an active locus without known biological functions and an AAV viral integration site on the human genome; therefore, it is considered a “safe harbor” for genome engineering. Here, we report that expansion and activation of B cells allowed up to 44-fold expansion in 7 days of culture (Figure 1). Electroporation of B cells showed a slight reduction of overall cell health (Figure 2A) at 24 h post-transfection. Scatter plot analysis of the CD19 marker (Figure 2B) showed up to 83% reduction in the edited cells (Figure 2C).

TIDE analysis of the chromatographs (Figure 3A) revealed that the % indels was similar to the % protein loss by flow cytometry (Figure 3B). Flow cytometry analysis of the KI experiment showed that the cells that received AAV vector (Figure 4), together with RNP, expressed up to 64% EGFP-positive cells (Figure 5A) and later displayed successful integration by junction polymerase chain reaction (PCR) (Figure 5B). Cell counts showed that all samples quickly recovered within 3 days post-engineering (Figure 5C).

Protocol

Leukapheresis samples from healthy donors were obtained from a local blood bank. All experiments described here were determined to be exempt for research by the Institutional Review Board (IRB) and were approved by the Institutional Biosafety Committee (IBC) at the University of Minnesota.

NOTE: All experiments were performed in compliance with the universal precaution for bloodborne pathogens, with sterile/aseptic techniques and proper biosafety level-2 equipment.

1. Prepare supplements for B-cell expansion medium

1. Reconstitute CpG oligonucleotide to a concentration of 1 mg/mL.
2. Reconstitute CD40 ligand (CD40L) to a concentration of 100 μg/mL.
3. Reconstitute recombinant human IL-10 (rhIL-10) to a concentration of 50 μg/mL.
4. Reconstitute recombinant human IL-15 (rhIL-15) to a concentration of 10 μg/mL.
   NOTE: Keep each supplement in small aliquots at -20 °C to -80 °C for up to 6 months.

2. Prepare basal medium

1. Combine B-cell basal medium with 5% (v/v) media supplement for in vitro immune cell expansion (e.g., CTS Immune Cell SR) and 1% (v/v) penicillin and streptomycin.
2. Filter-sterilize the basal medium using a 0.22 µm filter adaptor into a sterilized bottle.
3. Keep the basal medium at 4 °C for up to 1 month.

3. Prepare B-cell expansion medium

1. Transfer the required amount of the basal medium into a sterile container to culture the B cells at 5 × 10⁵ cells/mL.
2. Supplement the basal medium with 1 μg/mL CpG, 100 ng/mL CD40L, 50 ng/mL rhIL-10, and 10 ng/mL rhIL-15.
3. Filter the B-cell expansion medium using a 0.22 µm filter.
4. Equilibrate the B-cell expansion medium in the tissue culture incubator at 37 °C, 5% CO₂ with humidity for at least 30 min before use.
   NOTE: Prepare fresh B-cell expansion medium to use for one day. Do not prepare the B-cell expansion medium to use for multiple days. This media recipe encourages proliferation of memory B cells and activated primary human B cells.

4. Human B-cell purification and expansion

NOTE: Add 99–100% isopropyl alcohol to a temperature-controlled freezing container, following the manufacturer’s instruction, and chill the freezing container at 4 °C before starting step 4.1.

1. Isolate PBMCs from a leukaphoresis sample
   1. Transfer a leukaphoresis sample (approximate 8–10 mL) to a sterile 50 mL conical tube.
   2. Bring up the volume to 35 mL with sterile 1x phosphate-buffered saline (PBS).
   3. Carefully layer a 35 mL leukaphoresis sample on 15 mL of density-gradient medium.
   4. Centrifuge at 500 × g for 25 min without brake, remove the plasma layer without disturbing the buffy coat (PBMC layer), collect the PBMCs from the interface, and transfer to a new sterile 50 mL conical tube.
   5. Bring up the PBMCs to 50 mL with 1x PBS.
   6. Centrifuge at 500 × g for 5 min without brake. Remove the supernatant without disturbing the PBMC pellet, which may appear red.
   7. Add 7 mL of ammonium-chloride-potassium lysis buffer, pipette 3 times to mix well, and incubate at room temperature (RT) for 3 min.
   8. Bring up the volume to 50 mL with 1x PBS.
   9. Centrifuge at 400 × g for 5 min with low-resistance brake. Remove the supernatant without disturbing the pellet. The pellet should look pinkish or white.
      NOTE: To continue to culture the freshly isolated B cells, prepare the B-cell expansion medium before starting B-cell isolation (step 4.2).
2. B-cell isolation from PBMCs using human primary B-cell negative isolation kit
   1. Resuspend PBMCs in the isolation buffer to a concentration of 5 × 10⁷ cells/mL.
      NOTE: If the total number of PBMCs is less than 5 × 10⁷ cells, scale down the volume of isolation buffer to maintain 5 × 10⁷ cells/mL. Minimum and maximum volumes of cell suspension are 0.25 mL and 8 mL, respectively.
   2. Transfer up to 8 mL (5 × 10⁷ cells/mL) to a sterile, polypropylene, round-bottom tube with cap.
   3. Add 50 μL/mL of Cocktail Enhancer to the PBMCs.
   4. Add 50 μL/mL of Isolation Cocktail to the PBMCs, cap the tube, and invert 2–3 times to mix.
   5. Incubate at RT for 5 min; at the 4^th minute of incubation, vortex magnetic microbeads for at least 30 s.
   6. Transfer 50 μL of magnetic microbeads per 1 mL of PBMCs, cap the tube, and invert 2–3 times to mix.
   7. Top up to 10 mL with isolation buffer and gently pipette up and down 2–3 times.
   8. Place the tube in a magnetic station and incubate at RT for 3 min.
   9. Hold the magnet and tube together, and in one motion, invert the magnet and tube together to pour the cell suspension into the new tube. Discard the old tube.
   10. Repeat step 4.2.8 (reduce the incubation time to 2 min) and pour the B-cell suspension into a clean conical tube.
   11. The enriched B cells are ready to use. If cells will be used immediately, continue to section 4.3 (Human B-cell expansion). Check the purity of the isolated B cells by flow cytometry (optional). If cells are to be frozen before use, continue to step 4.2.12.
   12. To freeze the B cells, centrifuge at 400 × g for 5 min, and discard the supernatant without disturbing the pellet.
   13. Resuspend the cells in freezing medium at 10⁷ cells/mL, and aliquot 1 mL/cryovial.
   14. Place the cryovial in the chilled freezing container, and store at -80 °C overnight; then, transfer the frozen cryovial to a liquid nitrogen tank; keep frozen cells up to 1 year.
       NOTE: Expected yield of isolated B cells is 2%–8% of the total PBMCs, with 95%–99% viability.
3. Human B-cell expansion
   NOTE: If using freshly isolated B cells, skip steps 4.3.1–4.3.5. Count the cells and transfer the required number of cells into a sterile conical tube and continue with step 4.3.6.
   1. Pre-warm fetal bovine serum (FBS) in a water bath prior to thawing the B cells. Prepare 20 mL of B-cell expansion medium, transfer to a T25 flask, and pre-equilibrate the medium in a tissue-culture incubator (at 37 °C, 5% CO₂, with humidity) at least 15 min before use.
   2. Thaw B cells in a 37 °C water bath. While waiting, transfer 2 mL of pre-warmed FBS into a sterile 15 mL conical tube.
   3. After the B cells are completely thawed, immediately add 1 mL of pre-warmed FBS, drop-wise, into the sample. Incubate at RT for 1 min.
   4. Gently pipette to resuspend the sample and transfer the whole volume, dropwise, into a conical tube containing 2 mL of pre-warmed FBS.
   5. Bring up the volume to 15 mL with sterile 1x PBS, cap, and invert the tube gently 2–3 times.
   6. Centrifuge at 400 × g for 5 min.
   7. Discard the supernatant without disturbing the cell pellet, resuspend the cell pellet with 1 mL of pre-equilibrated B-cell expansion medium, and count the cells. The total cell number should be approximately 10⁷ cells.
   8. Transfer the cells into a flask containing 20 mL of the pre-equilibrated B-cell expansion medium. The final concentration of the cells should be approximately 5 x 10⁵ cells/mL.
   9. Incubate the flask vertically in a tissue-culture incubator.
   10. Refresh the expansion medium completely every 2 days by transferring the whole volume of B cells in to a sterile conical tube and repeat steps 4.3.6–4.3.9.
       NOTE: T25 flask can hold 10–20 mL of medium; T75 flask can hold up to 20–60 mL of medium.

5. Primary human B-cell engineering

1. Engineer B cells at 48 ± 2 h after expansion/activation for optimal results. Prepare the B-cell expansion medium, aliquot 1 mL of the medium into a 48-well tissue-culture plate, and pre-equilibrate in a tissue-culture incubator at least 15 min prior to use.
   NOTE: When designing a CRISPR/Cas9 sgRNA for a gene of interest, follow the steps outlined below.
   - Design sgRNAs using an online tool¹¹.
   - Design sgRNAs on an exon that is common to all isoforms of the protein.
   - Order chemically modified sgRNA from reputable companies.
   - sgRNA usually comes in lyophilized form; reconstitute sgRNA in sterile DNase/RNase-free Tris-EDTA (TE) buffer to a concentration of 1 μg/μL.
2. Prepare CRISPR/Cas9 transfecting substrate by mixing 1 μL (1 μg/μL) of chemically modified sgRNA with 1.5 μL (1 μg/μL) of chemically modified Streptococcus pyogenes Cas 9 (S.p. Cas9) nuclease per transfection reaction. For control, use 1 μL of TE buffer instead of sgRNA.
   NOTE: When CD19 sgRNA is used for a gene KO experiment, see Figure 2 for results. When AAVS1 sgRNA is used for a gene KI experiment, see Figure 5 for results.
   • Keep all the components on ice.
3. Mix gently and transfer 2.5 μL of CRISPR/Cas9 transfecting substrate per reaction into a tube of a 0.2 mL 8-tube strip; set aside at RT.
4. Turn on a nucleofector (electroporator), and prepare transfection reagents as shown in Table 1.
   NOTE: This is a good step for a pause, if necessary, by putting all reagents on ice. Remove all reagents from ice when ready to resume the experiment. When using S.p. Cas9 protein, the investigator MUST pre-complex CRISPR/Cas9-sgRNA ribonucleoprotein by mixing 1 μg sgRNA with 5 μg of S.p. Cas9 protein, avoiding any bubbles, and incubating the mixture at RT for at least 20 min before use for optimal results.
5. Count and transfer 10⁶ B cells per transfection reaction into a sterile conical tube.
6. Bring up the volume to 15 mL with sterile 1x PBS, and centrifuge at 400 × g for 5 min. While waiting, prepare the primary cell transfection reagent (Table 2) and set aside at RT.
7. Discard the supernatant without disturbing the cell pellet.
8. Resuspend the cell pellet with 10 mL of sterile 1x PBS and centrifuge at 400 × g for 5 min.
9. Discard the supernatant completely without disturbing the cell pellet.
10. Transfer 0.5 μg of chemically modified GFP mRNA (as a transfection reporter) per 10⁶ B cells to the cell pellet (optional).
11. Resuspend the cell pellet with 20 μL primary cell transfection reagent per 10⁶ B cells; mix gently by pipetting 5–6 times. Transfer 20.5 μL per transfection reaction into the 0.2 mL tube of the 8-tube strip containing 2.5 μL of the CRISPR/Cas9 transfection substrate.
12. Pipette up and down once to mix and transfer the entire volume (23 μL) into a transfection cuvette. Cap and tap the cuvette on the bench gently to ensure that the liquid covers the bottom of the cuvette.
13. Use human primary B-cell protocol on the nucleofector for transfection.
    NOTE: The nucleofector (electroporator) can be placed and be used outside the tissue-culture hood. Cap the cuvette to ensure sterility.
14. Rest the electroporated cells in the cuvette at RT for 15 min.
15. Transfer 80 μL of the pre-equilibrated B-cell expansion medium from the tissue culture plate into the transfection reaction in the cuvette. Place the cuvette in the tissue culture incubator for 30 min.
16. Gently pipette a couple times to mix and transfer the whole volume of the sample from the cuvette to an appropriate well of a 48-well tissue-culture plate containing 1 mL of the B-cell expansion medium. The final concentration of the cells should be 10⁶ cells/mL.
17. If performing a gene KI experiment, transfer rAAV6 vector at 500,000 multiplicity of infection into the appropriate well containing electroporated cells (approximately 45 min post-electroporation). See example rAAV6 vector construct in Figure 4A.
    NOTE: For example: A control sample will be electroporated without CRISPR/Cas9 or the rAAV6 vector. A vector-only sample will be electroporated without CRISPR/Cas9 and then be transduced with the rAAV6 vector. A KI sample will be electroporated with CRISPR/Cas9 and then be transduced with the rAAV6 vector. rAAV6 must contain homology arms up- and downstream of the targeted DSB site for HDR.
18. Place the plate in a tissue culture incubator at 37 °C and 5% CO₂ with humidity.
19. Count the cells and record viability at day 1 post-engineering.
20. Refresh the B-cell expansion medium every 2 days by counting the cells and then transferring the whole volume of the cells into a clean 1.5 mL microcentrifuge tube. Centrifuge at 400 × g for 5 min, and discard the supernatant without disturbing the pellet. Resuspend the cells with 100 µL of fresh B-cell expansion medium, and transfer to a well of a 24-well tissue culture plate. Bring up the medium volume to achieve a final cell concentration at 5 × 10⁵ cells/mL.
    NOTE: A 48-well plate can hold up to 1 mL medium/well; a 24-well plate can hold up to 2 mL medium/well; a 12-well plate can hold up to 4 mL medium/well, and a 6-well plate can hold up to 8 mL medium/well.
21. Allow the engineered cells to expand for at least 5 days before performing downstream analyses such as for flow cytometry analysis, TIDE analysis, and junction PCR.

Results

The updated expansion and activation protocol enabled the rapid expansion of B cells up to 44-fold in 7 days (Figure 1; n =3 donors). In the KO experiment, the B-cell count using Trypan blue staining showed more than 80% viable cells with a slight reduction in cell recovery in both the control and the CD19 KO samples at 24 h post-electroporation (Figure 2A; p ≥ 0.05, n = 3 donors). This result indicates that electroporation slightly affected overall B-cell...

Discussion

Precise genome engineering in primary human B cells has been challenging until recently^9,10. We had previously published protocols using CRISPR/Cas9 to engineer primary human B cells⁹. Here, we outline improved protocols for B-cell isolation, expansion, and engineering to allow for efficient KO of CD19 or for knocking-in EGFP.

Here we demonstrate that our expansion protocol allows the rapid expansion of...

Materials

Name	Company	Catalog Number	Comments
Alt-R S.p. Cas9 Nuclease V3 protein, 500 ug	IDT	1081059	smaller size is also available
Amaxa P3 primary cell 4D- Nucleofector X Kit S (32 RCT)	Lonza	V4XP-3032
Ammonium-chloride-potassium (ACK) lysing buffer	Quality Biological	118-156-101
CleanCap chemically modified Cas9 mRNA	Trilink Biotechnology	L-7206-1000
CpG ODN 2006 (ODN 7909) 5 mg	Invivogen	TLRL-2006-5	different sizes available
Cryostor CS10, 100 mL	STEMCELL Technology	7930
CTS Immune Cell SR	Thermo Fisher Scientific	A2596101
EasySep human B cells isolation kit	STEMCELL Technology	17954
eBioscience fixable viability dye eFlour 780	eBiosciences	65-0865-14
Excellerate B cell media, Xeno-free	R&D Systems	CCM031	B-cell basal medium
Falcon 14 mL Polypropylene Round-bottom Tube	Corning	352059
Fetal Bovine Serum (FBS)	R&D Systems	S11550	For thawing B cells only
Ficoll-Paque Plus	GE Healthcare	17-1440-03
GeneMate SnapStrip® 8-Strip 0.2 mL PCR Tubes with Individual Attached Dome Caps	BioExpress	T-3035-1 / 490003-692
Hyclone 0.0067M PBS (No Ca, No Mg) or 1x PBS	GE lifesciences	SH30256.01
Lonza 4D Nucleofector core unit	Lonza	AAF-1002B
Lonza 4D Nucleofector X unit	Lonza	AAF-1002X
Mega CD40 Ligand	Enzo Life Sciences	ALX-522-110-C010
Mr. Frosty	Sigma-Aldrich	C1562-1EA	For freezing cells
Pen/Strep 100X	Sigma-Aldrich	TMS-AB2-C
PerCP anti-human CD19 clone HIB19	biolegend	302228	smaller size is also available
rAAV6 SA-GFP pakaging ( with our SA-GFP cassette see Figure 4.)	Vigene Biosciences	N/A	large scale packaging, 1e13 GC/mL, 500 mL
Recombinant human IL-10 protein 250 ug	R&D Systems	217-IL-250	different sizes available
Recombinant human IL-15 protein 25 ug	R&D Systems	247-ILB-025	different sizes available
The Big Easy EasySep Magnet	STEMCELL Technology	18001	different sizes available
Tris-EDTA (TE) buffer	Fisher Scientific	BP2476100