I have attached my primary paper and paper direction below.
Here is the direction for the paper. Primary Research paper to be used is attached separately. 1) Length: 5 to 6 pages long 2) Typed (12 pt font), Double-spaced, and one-inch margins 3) Include separate Title and Reference pages. These do not contribute to the total page count. 4) References: Unless approved by me, web-based resources will not be accepted. a. Journal Article: Brophy, B., Smolenski, G., Wheeler, T., Wells, D., L'Huillier, P., and Laible, G. 2003. Cloned transgenic cattle produce milk with higher levels of -casein and -casein.Nature Biotechnology, 21(2):111-211. b. Book: Gershon, A., P. LaRussa, and S. Steinberg. 1999. Varicella-zoster virus, p.900-911. In P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H. Yolken(ed.), Manual of Clinical Microbiology, 7th Ed. American Society for Microbiology, Washington, D.C. c. When citing a reference, use the last names of authors and the year of the article. Examples: (Smith, 1995) for a single author (Smith and Jones, 1996) for two authors (Smith, et al., 1998) for more than two authors 5) You may include figures in your paper if you wish, but they are not required. Figures do not count as part of the paper’s length and should be included in an Appendix at the end of the paper. Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins Chung-An M. Wua,1, Theodore L. Rothb,1, Yuriy Baglaenkoh,i,1,3, Dario M. Ferrih,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 bDepartment 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 Zuckerberg 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-corresponding 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: 415-476-5178, Chris.Allen@ucsf.edu, 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: 415-502-2611, Alexander.Marson@ucsf.edu, Joan E. Wither, Krembil Research Institute, University Health Network, 60 Leonard Ave, Toronto, Ontario M5T 2S8, Canada, Tel: 416-603-5048, Joan.Wither@uhnresearch.ca. 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 customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 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.2018.03.009. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript iDepartment of Immunology, 60 Leonard Ave, University of Toronto, Toronto, Ontario, Canada jSunnybrook 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 research and therapeutic applications (1). The engineered nuclease CRISPR-Cas9 has revolutionized 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), correction of a CFTR gene defect in intestinal stem cell organoids cultured from cystic fibrosis 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, arrayed 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. Wu et al. Page 2 J Immunol Methods. Author manuscript; available in PMC 2019 June 01. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript 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 conferred 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 broad 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 blood 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