CRISPR/Cas9 technology has revolutionized genetic engineering of primary cells. Although its use is gaining momentum in studies on CD8⁺ T cell biology, it remains elusive to what extent CRISPR/Cas9 affects in vivo function of CD8⁺ T cells. Here, we optimized nucleofection-based CRISPR/Cas9 genetic engineering of naïve and in vitro-activated primary mouse CD8⁺ T cells and tested their in vivo immune responses. Nucleofection of naïve CD8⁺ T cells preserved their in vivo antiviral immune responsiveness to an extent that is indistinguishable from non-nucleofected cells, whereas in vitro activation of CD8⁺ T cells prior to nucleofection led to slightly impaired expansion/survival. Of note, different target proteins displayed distinct decay rates after gene editing. This is in stark contrast to a comparable period of time required to complete gene inactivation. Thus, for optimal experimental design, it is crucial to determine the kinetics of the loss of target gene product to adapt incubation period after gene editing. In sum, nucleofection-based CRISPR/Cas9 genome editing achieves efficient and rapid generation of mutant CD8⁺ T cells without imposing detrimental constraints on their in vivo functions.

中文翻译：

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在基于电穿孔的 CRISPR/Cas9 基因工程后，幼稚和体外激活的原代小鼠 CD8+ T 细胞保持体内免疫反应性

CRISPR/Cas9 技术彻底改变了原代细胞的基因工程。尽管其在 CD8 ⁺ T 细胞生物学研究中的应用势头强劲，但 CRISPR/Cas9 对CD8 ⁺ T 细胞体内功能的影响程度仍然难以捉摸。在这里，我们优化了初始和体外激活的原代小鼠 CD8 ⁺ T 细胞的基于核转染的 CRISPR/Cas9 基因工程，并测试了它们的体内免疫反应。幼稚 CD8 ⁺ T 细胞的核转染将其体内抗病毒免疫反应性保留到与非核转染细胞无法区分的程度，而CD8 的体外激活⁺核转染前的 T 细胞导致扩增/存活轻微受损。值得注意的是，不同的靶蛋白在基因编辑后显示出不同的衰减率。这与完成基因失活所需的可比时间形成鲜明对比。因此，为了优化实验设计，确定目标基因产物丢失的动力学以适应基因编辑后的潜伏期至关重要。总之，基于核转染的 CRISPR/Cas9 基因组编辑实现了高效快速的突变 CD8 ⁺ T 细胞生成，而不会对其体内功能施加不利限制。