Homo sapiens Targeted loci cultured

B cells differentiate into long-lived plasma cells that provide humoral immunity by secreting large quantities of antibodies. The ability to engineer primary human B cells to secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. To achieve this goal, we developed methods for efficient genome editing of primary B cells isolated from peripheral blood, followed by ex vivo differentiation into plasma cells. By delivering CRISPR/Cas9 ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates up to 94%). We first used this method to modulate plasma cell differentiation by disrupting key developmental regulatory genes. Next, we co-delivered RNPs with either single-stranded DNA oligonucleotide and achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.B细胞（B cells）可分化为长寿命浆细胞（long-lived plasma cells），通过分泌大量抗体介导体液免疫（humoral immunity）。对原代人B细胞（primary human B cells）进行工程化改造以分泌全新蛋白质的能力，可为蛋白质缺乏症及其他临床应用开发新型浆细胞疗法提供可行路径。为达成这一目标，我们开发了针对外周血分离获取的原代人B细胞的高效基因组编辑方法，随后可将其体外（ex vivo）诱导分化为浆细胞。通过在B细胞快速扩增的条件下递送CRISPR/Cas9核糖核蛋白（ribonucleoprotein, RNP）复合物，我们在原代人B细胞的多个基因组位点实现了位点特异性基因敲除，编辑效率最高可达94%。我们首先利用该方法，通过敲除关键发育调控基因来调控浆细胞分化过程。随后我们将核糖核蛋白复合物与单链DNA寡核苷酸（single-stranded DNA oligonucleotide）共递送，并通过同源定向修复（homology-directed repair）实现了高效的靶向序列整合，整合效率最高可达40%。本研究结果凸显了基因组编辑在人B细胞生物学研究中的应用价值，并证实了一种改造人浆细胞以分泌治疗性蛋白质的全新策略。