Multiplexed CRISPR/CAS9‐mediated engineering of pre‐clinical mouse models bearing native human B cell receptors. (1st December 2020)

• Record Type: Journal Article
• Authors: Wang, Xuesong; Ray, Rashmi; Kratochvil, Sven; Melzi, Eleonora; Lin, Ying‐Cing; Giguere, Sophie; Xu, Liling; Warner, John; Cheon, Diane; Liguori, Alessia; Groschel, Bettina; Phelps, Nicole; Adachi, Yumiko; Tingle, Ryan; Wu, Lin; Crotty, Shane; Kirsch, Kathrin H; Nair, Usha; Schief, William R; Batista, Facundo D
• Is Part Of: EMBO journal. Volume 40:Number 2(2021)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-12-01
• Subjects: antibody -- B cell receptor -- CRISPR -- Cas9 -- HIV vaccine -- knock‐in; Molecular biology -- Periodicals; 572.805
• DOI: 10.15252/embj.2020105926
• Languages: English
• ISSNs: 0261-4189

Abstract

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. Abstract : Application of a new one‐step genome editing protocol enables fast and efficient insertion of both immunoglobulin heavy and light chains from the human germline at their respective endogenous loci in mice.