Journal of Immunological Methods 457 (2018) 33–40 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim Research paper Genetic engineering in primary human B cells with CRISPR-Cas9 T ribonucleoproteins Chung-An M. Wua,1, Theodore L. Rothb,1, Yuriy Baglaenkoh,i,1,2, Dario M. Ferrih,i, ⁎⁎⁎ Patrick Braueri,j, Juan Carlos Zuniga-Pfluckeri,j, Kristina W. Rosbef, Joan E. Witherh,i,k, , ⁎⁎ ⁎ Alexander Marsonb,c,d,e, , Christopher D.C. Allena,g, a Cardiovascular Research Institute, Sandler Asthma Basic Research Center, 555 Mission Bay Blvd S, University of California, San Francisco, San Francisco, CA 94143, USA b Department of Microbiology and Immunology, 513 Parnassus Ave, University of California, San Francisco, CA 94143, USA c Department of Medicine, Diabetes Center, Helen Diller Family Comprehensive Cancer Center, 513 Parnassus Ave, University of California, San Francisco, CA 94143, USA d Innovative Genomics Institute, 2151 Berkeley Way, University of California, Berkeley, Berkeley, CA 94720, USA e Chan Zuckerberg Biohub, 499 Illinois St, San Francisco, CA 94158, USA f Department of Otolaryngology, 550 16th St, University of California, San Francisco, San Francisco, CA 94143, USA g Department of Anatomy, 555 Mission Bay Blvd S, University of California, San Francisco, San Francisco, CA 94143, USA h Krembil Research Institute, 60 Leonard Ave, University Health Network, Toronto, Ontario, Canada i Department of Immunology, 60 Leonard Ave, University of Toronto, Toronto, Ontario, Canada j Sunnybrook Research Institute, 2075 Bayview Ave, University of Toronto, Toronto, Ontario, Canada k Department of Medicine, 60 Leonard Ave, University of Toronto, Toronto, Ontario, Canada ARTICLE INFO ABSTRACT Keywords: Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic CRISPR-Cas9 genome engineering applications, but extension to primary human B cells remains limited. Here we report a Cas9 ribonucleoprotein method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 Primary human B cells ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies Genome engineering that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with effi- ciency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We de- livered 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. 1. Introduction relevant biology that may not be conserved in model organisms (Barrangou and Doudna, 2016). Recent studies also provide pre-clinical The ability to genetically manipulate human cells provides immense evidence for the potential of CRISPR in therapeutic applications, such opportunity for research and therapeutic applications (Lombardo and as disruption of the hepatitis B virus (Zhen et al., 2015), prevention of Naldini, 2014). The engineered nuclease CRISPR-Cas9 has re- muscular dystrophy via germline DNA editing in a mouse model (Long volutionized the ability to generate targetable knockout and knock-in et al., 2014), correction of a CFTR gene defect in intestinal stem cell genomic edits, facilitating mechanistic genetic studies directly in pri- organoids cultured from cystic fibrosis patients (Schwank et al., 2013), mary human cells, which is critical for understanding medically and skin transplantation of human epidermal progenitor cells ⁎ Correspondence to: 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. ⁎⁎ Correspondence to: 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. ⁎⁎⁎ Correspondence to: Joan E. Wither, Krembil Research Institute, University Health Network, 60 Leonard Ave, Toronto, Ontario M5T 2S8, Canada. E-mail addresses: [email protected] (J.E. Wither), [email protected] (A. Marson), [email protected] (C.D.C. Allen). 1 Authors contributed equally. 2 Present address: Building for Transformative Medicine, 60 Fenwood Rd., Brigham and Women's Hospital, Boston, MA 02115, USA. https://doi.org/10.1016/j.jim.2018.03.009 Received 10 January 2018; Received in revised form 24 March 2018; Accepted 26 March 2018 Available online 31 March 2018 0022-1759/ © 2018 Elsevier B.V. All rights reserved. C.-A.M. Wu et al. Journal of Immunological Methods 457 (2018) 33–40 engineered to secrete GLP-1 as a treatment for obesity in mice (Yue the addition of a 5′ BglII restriction site and a partial P2A linker at the 3′ et al., 2017). site, using the following primers: forward 5′-cg gaa ttc AGA TCT ATG The components of CRISPR-Cas9 can be delivered in multiple ways, ATC GAA ACA TAC AAC CAA ACT TC-3′; reverse 5′- C CTC CAC GTC including viral transduction. Electroporation of Cas9 ribonucleopro- TCC AGC CTG CTT CAG CAG GCT GAA GTT AGT AGC TCC GCT TCC teins (RNPs), comprised of synthetic guide RNA (gRNA) and Cas9 GAG TTT GAG TAA GCC AAA GG-3′. The BAFF coding region was protein, has emerged as a method for high efficiency editing in primary amplified using the partial P2A linker sequence at the 5′ site and a human T cells (Schumann et al., 2015). RNP assembly does not require BamHI site at the 3′ site using the following primers: forward 5′-CTG molecular cloning, which allows this approach to be readily scaled into CTG AAG CAG GCT GGA GAC GTG GAG GAG AAC CCT GGA CCT ATG a high-throughout, arrayed platform (Hultquist et al., 2016). In addi- GAT GAC TCC ACA GAA AGG-3′; reverse 5′-gcg tcg GGA TCC TCA CAG tion, electroporation obviates the need for viral production and stable CAG TTT CAA TGC AC-3′. Both products were purified and joined in a genomic integration of CRISPR components, thereby simplifying ex- PCR reaction using the flanking primers. The CD40L-P2A-BAFF product perimentation and offering potential safety benefits for eventual clin- and the pMIY2 retroviral expression construct with an IRES EYFP re- ical applications. porter downstream of the cloning site were digested using BglII and B cells present an attractive platform for genetic editing given their BamHI and then ligated, and the correct sequence was verified. Ret- involvement in numerous autoimmune and infectious diseases rovirus was generated by transfection of 293 T cells with Effectene (Granholm and Cavallo, 1992). One report described targeting of the (Qiagen) and a mixture of pMIY2-CD40L-P2A-BAFF and the GAG/Pol immunoglobulin heavy chain locus in order to enforce class switching and VSV packaging plasmids. The medium was replaced at 24 h and in mouse B cells and immortalized human-derived B cell lines (Cheong collected at 48, 72, and 96 h. The virus-containing medium was filtered et al., 2016), while another used targeted gene knockouts to study V(D) and then used to transduce low-passage OP9 bone marrow stromal cells J recombination in mouse pro-B cell lines (Lenden Hasse et al., 2017). (ATCC-CRL2749). OP9 cells that were positive for YFP and therefore Other studies have demonstrated the ability to generate high-efficiency infected with the retrovirus were stained with an anti-CD40L-PE anti- gene knockouts in primary mouse B cells expressing a Cas9 transgene body (Clone 24–31, Biolegend) were sorted using a FACS Aria II cell (Chu et al., 2016, 2017). Extension of these CRISPR-based editing sorter (BD). OP9 or OP9-BAFF/CD40L cells were cultured in Alpha- techniques to primary human B cells has clear applications. While most MEM media (Invitrogen) supplemented with 20% fetal bovine serum research studies of B cells have been conducted in model systems or cell (FBS, Wisent). lines, use of CRISPR could enable detailed molecular and mechanistic studies of primary human B cells, providing valuable new insights into 2.2. Human B cell isolation and culture molecular function that may be relevant to human disease. B cells have also received minimal attention as a platform for therapeutic genetic 2.2.1. Peripheral blood B cell isolation and culture manipulation, in contrast to T cells, of which engineered cell therapies Healthy human subjects between the ages of 18 and 40 years with are already clinically approved (Bach et al., 2017; Neelapu et al., 2017). no family history of autoimmune disease were recruited with approval Given the critical role of the B cell in humoral immunity, the vast range by the Research Ethics Board of the University Health Network and of potential peptide and non-peptide specificities conferred by the B cell informed consent of all subjects for collection of human peripheral receptor, and its ability to act at a distance via secretion of soluble blood samples. Peripheral blood mononuclear cells (PBMCs) were iso- immunoglobulin (LeBien and Tedder, 2008), engineered B cell thera- lated via Ficoll-Paque Plus (GE Healthcare) gradient centrifugation pies would have broad potential applications. following vendor instructions,