Barnes Christopher R, Lee Hyuncheol, Ojala David S, Lewis Kazuomori K, Limsirichai Prajit, Schaffer David V
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720, USA.
California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA.
Mol Ther Nucleic Acids. 2021 Jul 16;26:94-103. doi: 10.1016/j.omtn.2021.06.026. eCollection 2021 Dec 3.
We describe a genome-wide screening strategy to identify target genes whose modulation increases the capacity of a cell to produce recombinant adeno-associated viral (AAV) vector. Specifically, a single-guide RNA (sgRNA) library for a CRISPR-based genome-wide transcriptional activation screen was inserted into an AAV vector, and iterative rounds of viral infection and rescue in HEK293 producer cells enabled the enrichment of sgRNAs targeting genes whose upregulation increased AAV production. Numerous gain-of-function targets were identified, including spindle and kinetochore associated complex subunit 2 (SKA2) and inositol 1, 4, 5-trisphosphate receptor interacting protein (ITPRIP). Furthermore, individual or combinatorial modulation of these targets in stable producer cell lines increased vector genomic replication and loading into AAV virions, resulting in up to a 3.8-fold increase in AAV manufacturing capacity. Our study offers an efficient approach to engineer viral vector producer cell lines and enhances our understanding of the roles of SKA2 and ITPRIP in AAV packaging.
我们描述了一种全基因组筛选策略，以鉴定那些通过调节可提高细胞产生重组腺相关病毒（AAV）载体能力的靶基因。具体而言，将用于基于CRISPR的全基因组转录激活筛选的单向导RNA（sgRNA）文库插入AAV载体中，在HEK293生产细胞中进行多轮病毒感染和拯救，使得靶向那些上调可增加AAV产生的基因的sgRNA得以富集。鉴定出了许多功能获得性靶点，包括纺锤体和动粒相关复合体亚基2（SKA2）以及肌醇1,4,5-三磷酸受体相互作用蛋白（ITPRIP）。此外，在稳定的生产细胞系中对这些靶点进行单独或组合调节，可增加载体基因组复制以及装入AAV病毒颗粒的量，从而使AAV生产能力提高达3.8倍。我们的研究提供了一种工程化病毒载体生产细胞系的有效方法，并增进了我们对SKA2和ITPRIP在AAV包装中作用的理解。
