Bindu Dhanesh Sivadasan, Savage Justin T, Brose Nicholas, Bradley Luke, Dimond Kylie, Tan Christabel Xin, Eroglu Cagla
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
Department of Neonatology, Children's Mercy Hospital, Kansas City, MO 64108, USA.
bioRxiv. 2024 Oct 10:2023.01.17.524433. doi: 10.1101/2023.01.17.524433.
CRISPR/Cas9-based genome engineering enables rapid and precise gene manipulations in the CNS. Here, we developed a non-invasive astrocyte-specific method utilizing a single AAV vector, which we named GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/Cas9 System). We verified GEARBOCS' specificity to mouse cortical astrocytes and demonstrated its utility for three types of gene manipulations: knockout (KO); tagging (TagIn); and reporter knock-in (GeneTrap) strategies. Next, we deployed GEARBOCS in two test cases. First, we determined that astrocytes are a necessary source of the synaptogenic factor Sparcl1 for thalamocortical synapse maintenance in the mouse primary visual cortex. Second, we determined that cortical astrocytes express the synaptic vesicle associated Vamp2 protein and found that it is required for maintaining excitatory and inhibitory synapse numbers in the visual cortex. These results show that the GEARBOCS strategy provides a fast and efficient means to study astrocyte biology .
基于CRISPR/Cas9的基因组工程技术能够在中枢神经系统中实现快速且精确的基因操作。在此，我们开发了一种利用单一腺相关病毒载体的非侵入性星形胶质细胞特异性方法，我们将其命名为GEARBOCS（基于CRISPR/Cas9系统的星形胶质细胞基因编辑）。我们验证了GEARBOCS对小鼠皮质星形胶质细胞的特异性，并证明了其在三种基因操作类型中的实用性：基因敲除（KO）；标记（TagIn）；以及报告基因敲入（基因捕获）策略。接下来，我们在两个测试案例中应用了GEARBOCS。首先，我们确定星形胶质细胞是小鼠初级视觉皮层中丘脑皮质突触维持的突触生成因子Sparcl1的必要来源。其次，我们确定皮质星形胶质细胞表达与突触小泡相关的Vamp2蛋白，并发现它是维持视觉皮层中兴奋性和抑制性突触数量所必需的。这些结果表明，GEARBOCS策略为研究星形胶质细胞生物学提供了一种快速有效的方法。
