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Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins
Genetic engineering in primary human B cells with CRISPR-Cas9
ibonucleoproteins
Chung-An M. Wua,1, Theodore L. Rothb,1, Yuriy Baglaenkoh,i,1,3, Dario M. Fe
ih,i, Patrick
Braueri,j, Juan Carlos Zuniga-Pfluckeri,j, Kristina W. Rosbef, Joan E. Witherh,i,k,2, Alexander
Marsonb,c,d,e,2, and Christopher D.C. Allena,g,2
aCardiovascular Research Institute and Sandler Asthma Basic Research Center, 555 Mission Bay
Blvd S, University of California, San Francisco, San Francisco, CA 94143, USA
Department of Microbiology and Immunology, 513 Parnassus Ave, University of California, San
Francisco, CA 94143, USA
cDepartment of Medicine, Diabetes Center, and Helen Diller Family Comprehensive Cancer
Center, 513 Parnassus Ave, University of California, San Francisco, CA 94143, USA
dInnovative Genomics Institute, 2151 Berkeley Way, University of California, Berkeley, Berkeley,
CA 94720, USA
eChan Zucke
erg Biohub, 499 Illinois St, San Francisco, CA 94158, USA
fDepartment of Otolaryngology, 550 16th St, University of California, San Francisco, San
Francisco, CA 94143, USA
gDepartment of Anatomy, 555 Mission Bay Blvd S, University of California, San Francisco, San
Francisco, CA 94143, USA
hKrembil Research Institute, 60 Leonard Ave, University Health Network, Toronto, Ontario,
Canada
2Co-co
esponding authors: Christopher D.C. Allen, Cardiovascular Research Institute, Sandler Asthma Basic Research Center, and
Department of Anatomy, University of California, San Francisco, 555 Mission Bay Blvd S, San Francisco, CA 94143, USA, Tel:
XXXXXXXXXX, XXXXXXXXXX, Alexander Marson, Department of Microbiology and Immunology, Department of Medicine,
Diabetes Center, and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, 513 Parnassus Ave,
San Francisco, CA 94143, USA, Tel: XXXXXXXXXX, XXXXXXXXXX, Joan E. Wither, Krembil Research Institute,
University Health Network, 60 Leonard Ave, Toronto, Ontario M5T 2S8, Canada, Tel: XXXXXXXXXX, XXXXXXXXXX.
1Authors contributed equally
3Present address: Building for Transformative Medicine, 60 Fenwood Rd, Brigham and Women’s Hospital, Boston, MA 02115, USA
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
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Author Contributions
C-A.M.W., T.L.R., Y.B., D.M.F., P.B., J.C.Z-P., J.E.W., A.M., and C.D.C.A. designed research and analyzed data; C-A.M.W., T.L.R.,
and Y.B. performed research; J.E.W., A.M., and C.D.C.A. supervised research; K.W.R. provided tonsil samples; and C-A.M.W.,
T.L.R., Y.B., J.E.W., A.M., and C.D.C.A. wrote the manuscript.
Competing Financial Interests statement
The Allen and Wither labs declare no competing financial interests.
A.M. is a cofounder of Spotlight Therapeutics and serves as an advisor to Juno Therapeutics and PACT Therapeutics. The Marson lab
has received sponsored research support from Juno Therapeutics and Epinomics. Intellectual property has been filed on Cas9 RNP
delivery methods by the Marson lab.
HHS Public Access
Author manuscript
J Immunol Methods. Author manuscript; available in PMC 2019 June 01.
Published in final edited form as:
J Immunol Methods. 2018 June ; 457: 33–40. doi:10.1016/j.jim XXXXXXXXXX.
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iDepartment of Immunology, 60 Leonard Ave, University of Toronto, Toronto, Ontario, Canada
jSunny
ook Research Institute, 2075 Bayview Ave, University of Toronto, Toronto, Ontario,
Canada
kDepartment of Medicine, 60 Leonard Ave, University of Toronto, Toronto, Ontario, Canada
Abstract
Genome editing in human cells with targeted nucleases now enables diverse experimental and
therapeutic genome engineering applications, but extension to primary human B cells remains
limited. Here we report a method for targeted genetic engineering in primary human B cells,
utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout
mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further,
we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through
co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in
two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and
activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In
summary, we demonstrate a powerful and scalable research tool for functional genetic studies of
human B cell biology that may have further applications in engineered B cell therapeutics.
Keywords
CRISPR-Cas9; Cas9 ribonucleoprotein; primary human B cells; genome engineering
1. Introduction
The ability to genetically manipulate human cells provides immense opportunity for
esearch and therapeutic applications (1). The engineered nuclease CRISPR-Cas9 has
evolutionized the ability to generate targetable knockout and knock-in genomic edits,
facilitating mechanistic genetic studies directly in primary human cells, which is critical for
understanding medically-relevant biology that may not be conserved in model organisms (2).
Recent studies also provide pre-clinical evidence for the potential of CRISPR in therapeutic
applications, such as disruption of the hepatitis B virus (3), prevention of muscular
dystrophy via germline DNA editing in a mouse model (4), co
ection of a CFTR gene
defect in intestinal stem cell organoids cultured from cystic fi
osis patients (5), and skin
transplantation of human epidermal progenitor cells engineered to secrete GLP-1 as a
treatment for obesity in mice (6).
The components of CRISPR-Cas9 can be delivered in multiple ways, including viral
transduction. Electroporation of Cas9 ribonucleoproteins (RNPs), comprised of synthetic
guide RNA (gRNA) and Cas9 protein, has emerged as a method for high efficiency editing
in primary human T cells (7). RNP assembly does not require molecular cloning, which
allows this approach to be readily scaled into a high-throughout, a
ayed platform (8). In
addition, electroporation obviates the need for viral production and stable genomic
integration of CRISPR components, thereby simplifying experimentation and offering
potential safety benefits for eventual clinical applications.
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B cells present an attractive platform for genetic editing given their involvement in
numerous autoimmune and infectious diseases (9). One report described targeting of the
immunoglobulin heavy chain locus in order to enforce class switching in mouse B cells and
immortalized human-derived B cell lines (10), while another used targeted gene knockouts
to study V(D)J recombination in mouse pro-B cell lines (11). Other studies have
demonstrated the ability to generate high-efficiency gene knockouts in primary mouse B
cells expressing a Cas9 transgene (12, 13). Extension of these CRISPR-based editing
techniques to primary human B cells has clear applications. While most research studies of
B cells have been conducted in model systems or cell lines, use of CRISPR could enable
detailed molecular and mechanistic studies of primary human B cells, providing valuable
new insights into molecular function that may be relevant to human disease. B cells have
also received minimal attention as a platform for therapeutic genetic manipulation, in
contrast to T cells, of which engineered cell therapies are already clinically approved (14,
15). Given the critical role of the B cell in humoral immunity, the vast range of potential
peptide and non-peptide specificities confe
ed by the B cell receptor, and its ability to act at
a distance via secretion of soluble immunoglobulin (16), engineered B cell therapies would
have
oad potential applications.
To achieve genetic manipulation of primary human B cells, we developed a methodology to
deliver CRISPR-Cas9 RNPs by electroporation to B cells isolated from human peripheral
lood or tonsils. We demonstrated genetic editing in experimental conditions reflecting a
wide range of biological B cell states via application to two distinct in vitro culture systems,
one which retained B cells in an undifferentiated state via co-culture with feeder cell lines,
and another which permitted analysis of differentiating B cells that had been activated with
soluble factors. We ablated single or even multiple genes at once by delivering appropriately
targeted RNPs, and we additionally confirmed efficient editing at both genomic and protein
expression levels. Finally, we demonstrated knock-in editing of a targeted gene by