Abstract

Resource1 December 2020Open Access Transparent process Multiplexed CRISPR/CAS9-mediated engineering of pre-clinical mouse models bearing native human B cell receptors Xuesong Wang The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USAThese authors contributed equally to this work Search for more papers by this author Rashmi Ray The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USAThese authors contributed equally to this work Search for more papers by this author Sven Kratochvil The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Eleonora Melzi orcid.org/0000-0002-1520-735X The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Ying-Cing Lin The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Sophie Giguere The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Liling Xu The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author John Warner The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Diane Cheon The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Alessia Liguori Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Bettina Groschel Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Nicole Phelps Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Yumiko Adachi Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Ryan Tingle Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Lin Wu Genome Modification Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Shane Crotty Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA, USA Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Kathrin H Kirsch orcid.org/0000-0003-1532-1715 The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Usha Nair orcid.org/0000-0002-1927-6889 The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author William R Schief Corresponding Author [email protected] orcid.org/0000-0002-1120-0150 The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Facundo D Batista Corresponding Author [email protected] orcid.org/0000-0002-1130-9463 The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Department of Immunology, Harvard Medical School, Boston, MA, USA Department of Microbiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Xuesong Wang The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USAThese authors contributed equally to this work Search for more papers by this author Rashmi Ray The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USAThese authors contributed equally to this work Search for more papers by this author Sven Kratochvil The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Eleonora Melzi orcid.org/0000-0002-1520-735X The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Ying-Cing Lin The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Sophie Giguere The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Liling Xu The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author John Warner The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Diane Cheon The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Alessia Liguori Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Bettina Groschel Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Nicole Phelps Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Yumiko Adachi Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Ryan Tingle Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Lin Wu Genome Modification Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Shane Crotty Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA, USA Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Kathrin H Kirsch orcid.org/0000-0003-1532-1715 The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author Usha Nair orcid.org/0000-0002-1927-6889 The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Search for more papers by this author William R Schief Corresponding Author [email protected] orcid.org/0000-0002-1120-0150 The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Facundo D Batista Corresponding Author [email protected] orcid.org/0000-0002-1130-9463 The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA Department of Immunology, Harvard Medical School, Boston, MA, USA Department of Microbiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Xuesong Wang1, Rashmi Ray1, Sven Kratochvil1, Eleonora Melzi1, Ying-Cing Lin1, Sophie Giguere1, Liling Xu1, John Warner1, Diane Cheon1, Alessia Liguori2,3,4, Bettina Groschel2,3,4, Nicole Phelps2,3,4, Yumiko Adachi2,3,4, Ryan Tingle2,3,4, Lin Wu5, Shane Crotty4,6,7, Kathrin H Kirsch1, Usha Nair1, William R Schief *,1,2,3,4 and Facundo D Batista *,1,8,9 1The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA 2Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA 3IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA 4Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA, USA 5Genome Modification Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA 6Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA, USA 7Department of Medicine, University of California, San Diego, La Jolla, CA, USA 8Department of Immunology, Harvard Medical School, Boston, MA, USA 9Department of Microbiology, Harvard Medical School, Boston, MA, USA *Corresponding author. Tel: +1 858 784 7725; E-mail: [email protected] *Corresponding author. Tel: +1 857 268 7071; E-mail: [email protected] EMBO J (2021)40:e105926https://doi.org/10.15252/embj.2020105926 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract B-cell receptor (BCR) knock-in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one-step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD-GT8 60mer, a germline-targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class-switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases. Synopsis We report a one-step CRISPR/Cas9-mediated strategy to knock in rearranged human VDJ and VJ at endogenous mouse loci. The resulting B cells are functional: they undergo germinal center reactions, somatic hypermutations and differentiate into memory B cells. Generated knock-in mice bearing genuine human germline BCRs using a one-step CRISPR/Cas9-mediated strategy. B cells from mice bearing VRC01-class human BCRs are functional. Physiologically rare levels of VRC01-class precursors can be primed with eOD-GT8 60mer and undergo germinal center responses. VRC01-class precursors in adoptive transfer system undergo somatic hypermutations and exhibit VRC01-class mutations after a single priming injection. Introduction Humoral immunity defends against infections and plays a key role in immunization via the secretion of protective antibodies and production of long-lived memory B cells. The elicitation of protective antibodies through vaccination is dependent on a broad range of immunological principles, including: the activation of naïve B cells, their entry into germinal centers (GCs), affinity maturation of the B-cell receptors (BCRs) by iterative somatic hypermutation (SHM) dependent on T follicular cell help (TFH) within GCs, and ultimately the differentiation of GC B cells into long-lived memory B cells (MBCs) that can be recalled upon antigen re-exposure, and plasma cells (PCs) that secrete antibodies (Alt et al, 1992; Rajewsky, 1996; McHeyzer-Williams & McHeyzer-Williams, 2005; Vinuesa et al, 2005; Neuberger, 2008; Cyster, 2010; Crotty, 2011; Victora & Nussenzweig, 2012; Crotty, 2014). Although we draw upon these principles in order to successfully generate vaccines, a deeper understanding of the mechanisms that determine antibody specificity, diversification, affinity maturation, and durability is important in developing vaccines against antigenically diverse viruses such as HIV-1 and influenza (Burton et al, 2012; Haynes & Mascola, 2017). A key aim for vaccines against antigenically diverse viruses is to induce broadly neutralizing antibodies (bnAbs) capable of neutralizing diverse isolates. In the case of HIV-1, bnAbs develop in a small fraction of infected individuals, and passive transfer of bnAbs can prevent infection in animal models (Gauduin et al, 1997; Mascola et al, 2000; Parren et al, 2001; Moldt et al, 2012; Pietzsch et al, 2012; Shingai et al, 2014). Hence, it is widely accepted that vaccine induction of bnAbs with sufficient potency, breadth, and durability has potential to protect humans against HIV-1 (Burton et al, 2012; Burton & Hangartner, 2016; Kwong & Mascola, 2018; Haynes et al, 2019). However, while bnAbs bind HIV Env with high affinity, their unmutated precursors typically lack measurable affinity for most Env isolates. Germline-targeting vaccine design aims to alleviate this problem by engineering immunogens with affinity for unmutated bnAb precursors, which could initiate bnAb generation in the HIV seronegative human population (Jardine et al, 2013; McGuire et al, 2013; Dosenovic et al, 2015; Jardine et al, 2015; Briney et al, 2016; Escolano et al, 2016; McGuire et al, 2016; Sok et al, 2016; Steichen et al, 2016; Tian et al, 2016; Jardine et al, 2016a; Jardine et al, 2016b; Steichen et al, 2019). Pre-clinical testing of germline-targeting immunogens has often relied on knock-in (KI) mouse models expressing inferred germline (iGL) reverted precursors of known bnAbs (Dosenovic et al, 2015; Jardine et al, 2015; Briney et al, 2016; Escolano et al, 2016; McGuire et al, 2016; Steichen et al, 2016; Tian et al, 2016; Andrabi et al, 2019; Saunders et al, 2019; Steichen et al, 2019). While this approach has significantly contributed to the field and yielded several important insights, predicting the germline sequence of a highly mutated bnAb in the absence of longitudinal bnAb lineage sequencing data is challenging, in particular for the non-templated regions of the BCR. Furthermore, this raises the question of whether native, human BCRs will respond to germline-targeting Env immunogens in the same manner as iGLs. Recent studies in which the germline-targeting immunogen eOD-GT8 60mer was successfully employed as a bait to isolate native human B-cell precursors of the VRC01-class bnAbs family from HIV-1 seronegative volunteers provide the framework for addressing the question (Jardine et al, 2016a; Havenar-Daughton et al, 2018a; Havenar-Daughton et al, 2018b). VRC01-class bnAbs target the CD4-binding site of HIV-1 Env and include some of the broadest and most potent bnAbs that have been identified to date (Wu et al, 2010; Scheid et al, 2011; Wu et al, 2011; Huang et al, 2016; Sajadi et al, 2018). VRC01-class antibodies rely on a heavy chain (HC) using the IgHV1-2 gene, which structurally mimics CD4, a short 5-amino acid LCDR3 motif, and a flexible LCDR1, which is important to avoid a steric clash with gp120 (Zhou et al, 2010; Zhou et al, 2015). However, these Abs can accommodate a variety of different CDRH3s and LC V-genes, including Vκ3-20, Vκ1-33, Vκ1-5, and VL2-14 (Havenar-Daughton et al, 2018b; Umotoy et al, 2019). Seminal work carried out with earlier versions of the VRC01-class germline-reverted HC mouse models have revealed several important insights regarding the ability of tailored immunogens to drive affinity maturation (Dosenovic et al, 2015; Jardine et al, 2015; Briney et al, 2016). However, these studies did not initially address VRC01-class responses at physiologically relevant B-cell precursor frequencies. More recently, these issues have been overcome by taking advantage of a congenic adoptive transfer model, allowing experiments to be performed at well-defined precursor frequencies and affinities imparting relevance to the pre-clinical mouse models (Abbott et al, 2018; Dosenovic et al, 2018). However, a hurdle that now remains to be overcome is the time required for KI mouse generation. The mouse models generated thus far have exclusively relied on traditional embryonic stem (ES) cell technology, which suffers from the drawback that multiple crossings are required for the full germline transmission of insertions or deletions. To alleviate this barrier, we previously developed a one-step CRISPR/Cas9-induced homology-directed repair (HDR) approach. We generated mouse models bearing human, pre-rearranged Ig precursors for two different HIV bnAbs (PGT121gH or BG18gH) integrated at the endogenous mouse Ig H locus, in a matter of weeks (Lin et al, 2018; Steichen et al, 2019). In the PGT121gH KI mouse we observed a lack of PGT121 HC bearing B cells in the peripheral blood, indicating that B cells bearing PGT121gH may be auto- or poly-reactive in mice (Lin et al, 2018). In contrast, we found that BG18gH precursors could be stimulated after immunization; they accumulated SHM and gave rise to specific antibody responses (Steichen et al, 2019). However, at that point we were only successful in generating Ig H chain KI models, wherein the KI H chains paired with mouse Ig L chains. Previously, BCR KI mice expressing bicistronic Ig H and L chains targeted to the Ig H locus had been generated, but a rapid method to generate KI mice expressing Ig L chains at the Ig κ locus does not exist (Jacobsen et al, 2018). Therefore, in order to render the Ig KI model physiologically relevant, we extended this approach to generate Ig L chains at the mouse κ locus. Here, we report for the first time a protocol for the rapid generation of Ig L chains in the Igκ locus. Moreover, we extended this one-step CRISPR/Cas9 technology to multiplex the insertion of human germline Ig H and L chains at the Ig H and Ig κ native mouse loci, respectively. This protocol relies on the concomitant insertion of two large pieces of donor DNA at desired genomic loci of fertilized zygotes at high frequencies. We demonstrate the power of this technology by generating three novel KI mouse models expressing genuine human VRC01-class precursors isolated from healthy human donors (Havenar-Daughton et al, 2018b). We show that B cells expressing these VRC01-class BCRs can be primed in vivo by eOD-GT8 60mer, recruited to germinal centers even at rare precursor frequency, secrete class-switched antibodies, and undergo somatic hypermutation. Thus, these KI mice are highly valuable in evaluating immune response elicited by vaccine more authentically. It is a major step forward that not only reduces the time for HIV pre-clinical validation but is also a promising way to accelerate vaccine design for HIV based on the prime-boost strategy. Results Generation of KI mice expressing human light chain in one-step using CRISPR/Cas9 We have previously shown that using the CRISPR/Cas9 technology, we can accelerate the generation of KI mice expressing pre-rearranged, human germline-reverted Ig H chains at the native Ig H locus, via donor template-mediated homology-directed repair (HDR) zygote microinjection (Lin et al, 2018; Steichen et al, 2019). However, since BCR specificity is not conferred by the heavy chain alone, in this work we attempted to investigate if we can extend this methodology to generate KI mice expressing similar Ig L chains at the mouse κ locus. Indeed, the rapid generation of mouse models with Ig H and L chains knocked into their native loci would not only facilitate the generation of proper BCR specificity, but also allow the interrogation of class switch recombination, somatic hypermutation (SHM), and affinity maturation in response to infection and immunization under physiological conditions. We constructed donor plasmids with 3.9 kb 5ʹ and 3ʹ homology arms, C57/BL6 mouse Vκ4-53 promoter and its related leader region (1.5 kb), as well as a pre-assembled L chain (0.4 kb) expressing PGT121-gL, the predicted germline VJ sequence of the PGT121 monoclonal antibody light chain (Fig 1A). This sequence, previously described as PGT121 GLCDR3rev1 light chain, has been fully germline-reverted (Steichen et al, 2016). In order to introduce our KI DNA target in the correct genomic locus and drive homologous recombination, our strategy relied on using Cas9 nuclease to introduce double-stranded breaks specifically at the Jκ1-Jκ5 region of the native Igκ locus of fertilized oocytes. We initiated double-stranded breaks using two single guide RNAs (sgRNAs) as indicated in Fig 1A. We used the CRISPR DESIGN database (http://https://zlab.bio/guide-design-resources/) to design-specific guide RNAs to cleave precise regions of the native murine Igκ locus of fertilized oocytes. We tested 12 sgRNAs and selected two sgRNAs, sgRNA11 and sgRNA18, based on their efficiency to cleave a PCR amplicon containing the wild type (WT) genomic DNA target in an in vitro assay (Fig 1B). An additional reason for selecting these sgRNAs was that the CRISPR DESIGN database predicted minimal off-target effects on related genes. The selected sgRNAs, Cas9 protein, and template plasmid were injected into fertilized oocytes, which were subsequently implanted into pseudo-pregnant female mice. Thirty founder mice (F0) were born, of which seven carried the PGT121 κ chain insertion (23.3% success rate) by genotyping using TaqMan probes (Fig 1A and Appendix Fig S1A, Appendix Tables S1 and S2). Of the seven F0 founders, in six of them the PGT121 κ chain insertion was present in one Ig κ allelic locus, whereas the other Ig κ locus was wild type (κPGT121/WT); in the last animal (F0–15), the PGT121 κ chain was inserted in one Ig κ allelic locus and the WT Jκ1-Jκ5 segment was deleted in the other allele (Appendix Table S2). We further crossed all of our positive light chain KI F0 founders with WT mice and followed the frequency of germline transmission of the KI light chain. Although the numbers of F1 progeny were not large, four out of seven F0 mice appeared to transmit the KI PGT121 κ chain in a Mendelian fashion. Figure 1. Generation of a KI mouse model bearing a pre-rearranged PGT121 κ Strategy for the insertion of PGT121 pre-rearranged VJ into the mouse Ig κ locus. Targeting DNA donor with 5ʹ (3.9 kb) and 3ʹ (3.9 kb) homology arms to the C57BL/6 WT mouse Ig κ locus, murine promoter, leader, and the human PGT121 light chain VJ sequences are located between two homology arms. Two sgRNAs, 18 and 11, were targeted at J4-J5 region of Ig κ locus. CRISPR/Cas9-mediated HDR leads to the insertion of the promoter and PGT121 sequences into the C57BL/6 mouse genome. V segments, enhancers, and the kappa constant regions are shown in gray and labeled appropriately. Yellow rectangles represent J segments; dark blue oval represents the Vκ4-53 promoter (P); light blue line represents the inserted segment and red rectangles show the rearranged PGT121 VJ. "T" represents TaqMan probe. WT probes were used for the detection of WT allele, Leader probes were used for the detection of the 5ʹend of the insertion, the specific probes were used for the detection of pre-arranged VJ insertion for PGT121 (probe sequences, see Appendix Table S1). A fragment of genomic DNA (2.2 kb) was amplified by PCR and in vitro sgRNA-guided Cas9-mediated cleavage assay was performed with each of the sgRNAs. sgRNA-targeting sites are indicated by arrows, genomic DNA size is indicated by asterisk. B220+ single B cells from peripheral blood of three PGT121 LC KI naïve mice were sorted. B220+ B-cell populations and their frequencies are shown in FACS plots (left panel). Ig light chains from single-cell sorted B cells were PCR amplified and sequenced. The resulting IGLV libraries were compared to the PGT121 LC reference sequence. The pie charts indicate the frequency of IGLV sequences identical to human PGT121 (red) and mouse IGLV (gray). Download figure Download PowerPoint Next, to confirm the expression of the PGT121 κ chain within the cell-surface BCR, we isolated and single-cell sorted B220+ peripheral B cells (Live/CD4− CD8− Gr1− F4/80− B220+) from three F0 κPGT121/WT mice, and determined the frequency of PGT121 κ chain germline sequences via BCR sequencing. In these three κPGT121/WT animals, ~ 90.5% (57 out of 63) of the κ chain sequences were identical to the original PGT121 κ chain germline sequence, whereas ~ 9.5% (6 out of 63) were wild type (Fig 1C). We also obtained single-cell paired H-L chain sequences from B220+ peripheral B cells of the three F1 κPGT121/WT mice. We observed that PGT121 κ chain was paired with mouse Ig HC, suggesting that the PGT121 κ chain was part of a functional BCR. Furthermore, the Ig H V gene family usage also appeared to be more or less evenly distributed, and not skewed toward any one particular Ig H V gene (Appendix Fig S1B). Our results reveal that using the CRISPR/Cas9 technology, we are able to knock in a relatively large DNA fragment (~ 2 kb) containing the pre-rearranged VJ kappa to generate F0 animals in a matter of weeks, and that the knock-in allele is transmitted to the F1 progeny. In this test case, the CRISPR/Cas9-mediated generation of light chain KI mice was rapid and reliable. One-step KI mice generation expressing genuine human germline BCRs by multiplexing H and L chain in zygotes After establishing that we can individually rapidly generate H or L chain KI mice, we wanted to determine if we would be able to obtain the simultaneously insert pre-rearranged Ig H and L inserts into their respective native loci via CRISPR-mediated injection with two donor plasmids. The advantage of multiplexed HDR knock-in of H and L chains is that it will eliminate extra crosses between mice expressing H and L chains alone and save a considerable amount of time; however, the generation of double knock-in model is also a challenge. Indeed, the insertion of two large constructs at precise and native loci by zygote microinjection has not been reported so far. In order to test this notion, we attempted to generate KI mice expressing paired CLK21 H and L chains, which constitute one of the native human germline VRC01-class BCRs identified from a HIV-1 seronegative volunteer (Havenar-Daughton et al, 2018b). To expedite the generation of