Zhang Xiujuan, Hao Siyuan, Feng Zehua, Ning Kang, Kuz Cagla Aksu, McFarlin Shane, Richart Donovan, Cheng Fang, Zhang-Chen Ander, McFarlane Richenda, Yan Ziying, Qiu Jianming
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242, USA.
bioRxiv. 2024 Oct 17:2024.10.16.618764. doi: 10.1101/2024.10.16.618764.
We conducted a genome-wide CRISPR/Cas9 screen in suspension 293-F cells transduced with rAAV5. The highly selected genes revealed after two rounds of screens included the previously reported , , and , along with a cluster of genes involved in glycan biogenesis, Golgi apparatus localization and endoplasmic reticulum penetration. In this report, we focused on solute carrier family 35 member A1 (), a Golgi apparatus-localized cytidine 5'-monophosphate-sialic acid (CMP-SIA) transporter. We confirmed that knockout (KO) significantly decreased rAAV5 transduction to a level lower than that observed in or KO cells. Although KO drastically reduced the expression of α2,6-linked SIA on the cell surface, the expression of α2,3-linked SIA, as well as the cell binding and internalization of rAAV5, were only moderately affected. Moreover, KO significantly diminished the transduction of AAV multi-serotypes, including rAAV2 and rAAV3 which do not utilize SIAs for primary attachment. Notably, the SLC35A1 KO markedly increased transduction of rAAV9 and rAAV11, which primarily attach to cells via binding to galactose. Further analyses revealed that KO significantly decreased vector nuclear import. More importantly, although the C-terminal cytoplasmic tail deletion (ΔC Tail) mutant of SLC35A1 did not drastically decrease SIA expression, it significantly decreased rAAV transduction, as well as vector nuclear import, suggesting the C-tail is critical in these processes. Furthermore, the T128A mutant significantly decreased SIA expression, but still supported rAAV transduction and nuclear import. These findings highlight the involvement of the CMP-SIA transporter in the intracellular trafficking of rAAV vectors post-internalization.
我们在用rAAV5转导的悬浮293-F细胞中进行了全基因组CRISPR/Cas9筛选。两轮筛选后高度富集的基因包括先前报道的基因，以及一组参与聚糖生物合成、高尔基体定位和内质网穿透的基因。在本报告中，我们重点研究了溶质载体家族35成员A1（SLC35A1），一种定位于高尔基体的胞苷5'-单磷酸-唾液酸（CMP-Sia）转运蛋白。我们证实，SLC35A1基因敲除（KO）显著降低了rAAV5转导，降至低于在其他基因敲除细胞中观察到的水平。虽然SLC35A1基因敲除大幅降低了细胞表面α2,6连接的Sia的表达，但α2,3连接的Sia的表达以及rAAV5的细胞结合和内化仅受到中度影响。此外，SLC35A1基因敲除显著降低了包括rAAV2和rAAV3在内的多种AAV血清型的转导，rAAV2和rAAV3并不利用Sia进行初始附着。值得注意的是，SLC35A1基因敲除显著增加了rAAV9和rAAV11的转导，rAAV9和rAAV11主要通过与半乳糖结合附着于细胞。进一步分析表明，SLC35A1基因敲除显著降低了载体的核输入。更重要的是，虽然SLC35A1的C末端胞质尾缺失（ΔC尾）突变体并未大幅降低Sia表达，但它显著降低了rAAV转导以及载体核输入，表明C尾在这些过程中至关重要。此外，T128A突变体显著降低了Sia表达，但仍支持rAAV转导和核输入。这些发现突出了CMP-Sia转运蛋白在rAAV载体内化后细胞内运输中的作用。
