Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.

中文翻译：

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高效CRISPR-Cas9介导的基因敲除人类原代B细胞中的功能基因研究爱泼斯坦-巴尔病毒感染

现在，在所有原核和后生细胞中，基因编辑都是常规的，但是不到十年前，当CRISPR-Cas9技术被引入哺乳动物细胞生物学领域时，其在免疫细胞中并未受到太多关注。这种通用技术已经成功地应用于人类骨髓细胞和T细胞的基因修饰，但是对于人类原代B细胞的应用却很少，而且仅限于活化的B细胞。这种局限性使得无法对该类型细胞进行细胞活化，分化或细胞周期控制的结论性研究。我们报道了使用Cas9核糖核蛋白复合物的核转染在原代静息的人类B细胞中高效，简单和快速的基因组工程。我们提供使用两个模型基因：CD46和CDKN2A在静止的人B细胞中进行基因编辑的原理证明。后者编码细胞周期调节子p16INK4a，它是爱泼斯坦-巴尔病毒（EBV）的重要靶标。野生型和EBNA3癌蛋白突变株感染携带CDKN2A基因敲除的B细胞，从而使我们得出结论，EBNA3C控制CDKN2A，这是EBV感染细胞中B细胞增殖的唯一障碍。总之，这种方法能够有效地靶向静止的人类B细胞中的特定基因位点，从而支持基础研究以及免疫治疗策略。