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 m....

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 ....

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