Table2_CRISPR library screening to develop HEK293-derived cell lines with improved lentiviral vector titers.XLSX

Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers. Briefly, LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) were used to modify HEK293 and HEK293T cells. These cell populations were then expanded, and integrated LV vector genomes were rescued by transfection. LV vectors were harvested, and the process of sequential transduction and rescue-transfection was iterated. Through this workflow, guide RNAs (gRNAs) that target genes that may suppress or enhance LV vector production were enriched and identified with Next-Generation Sequencing (NGS). Though more work is needed to test genes identified in this screen, we expect that perturbations of genes we identified here, such as TTLL12, which is an inhibitor of antiviral innate immunity may be introduced and multiplexed to yield cell lines with improved LV vector productivity.

慢病毒载体（LV）已成为治疗遗传性疾病和获得性疾病的有力工具。随着临床研究和商业需求的不断进步，对大量纯化LV载体的需求日益增长。为满足这一需求，我们开发了基于CRISPR的筛选方法，以识别可能增加LV载体滴度的人类胚胎肾脏293（HEK293）细胞及其衍生物中的遗传变异。简而言之，我们使用了基于LV载体的Human CRISPR激活和敲除文库（Calabrese和Brunello），以改造HEK293和HEK293T细胞。随后，这些细胞群体得到扩增，并通过转染挽救了整合的LV载体基因组。收集LV载体后，我们通过迭代转导和挽救转染的过程。通过这一工作流程，利用下一代测序（NGS）富集和识别了靶向可能抑制或增强LV载体生产的基因的引导RNA（gRNAs）。尽管还需要更多的研究来验证筛选中确定的基因，但我们预期，我们在此识别的基因变异，如TTLL12（一种抗病毒先天免疫抑制因子），可通过引入和复用来产生具有改进LV载体生产力的细胞系。