GEARBOCS: An Adeno Associated Virus Tool for<i>In Vivo</i>Gene Editing in Astrocytes

Bindu, Dhanesh Sivadasan; Tan, Christabel Xin; Savage, Justin T; Eroğlu, Çağla

doi:10.1101/2023.01.17.524433

Cited by 2 publications

(2 citation statements)

References 91 publications

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“…Because constitutive loss of TSP1/2 impaired performance during high-effort instrumental tasks, we tested whether this behavior deficit can be attributed to the loss of TSP1/2, specifically in adult astrocytes. To ablate TSP1 and TSP2 in astrocytes, we utilized a viral approach involving adeno-associated viruses (AAVs) [96]. This astrocytespecific CRISPR-based gene-editing tool called GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/HITI System) uses a single AAV vector to knock out a gene in mice expressing Cas9 in a Cre recombinase-dependent manner [96].…”

Section: Conditional Deletion Of Tsp1/2 In Adult Astrocytes Does Not ...mentioning

confidence: 99%

“…To ablate TSP1 and TSP2 in astrocytes, we utilized a viral approach involving adeno-associated viruses (AAVs) [96]. This astrocytespecific CRISPR-based gene-editing tool called GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/HITI System) uses a single AAV vector to knock out a gene in mice expressing Cas9 in a Cre recombinase-dependent manner [96]. Using this methodology, we generated AAV vectors carrying guide RNAs (sgRNA) targeting Thbs1 promoter.…”

Section: Conditional Deletion Of Tsp1/2 In Adult Astrocytes Does Not ...mentioning

confidence: 99%

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Astrocytic thrombospondins 1 and 2 are required for cortical synapse development controlling instrumental performance

Lawal,

Ulloa Severino,

Wang

et al. 2024

Preprint

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During development, controlled synaptogenesis is required to form functioning neural circuits that underlie cognition and behavior. Astrocytes, a major glial-cell type in the central nervous system (CNS), promote synapse formation by secreting synaptogenic proteins. Thrombospondins 1 and 2 (TSP1/2), which act through their neuronal receptor α2δ-1, are required for proper intracortical excitatory synaptogenesis. In the adult brain, the loss of α2δ-1 impairs training-induced excitatory synaptogenesis in the anterior cingulate cortex (ACC), and this impairment leads to increased effort-exertion during high-effort tasks. Here, we tested whether TSP1 and TSP2 are required for controlling effort during operant conditioning by using a lever press for food reward training in mice. Surprisingly, we found that constitutive loss of TSP1/2 significantly reduced lever pressing performance when the effort required for a food reward was increased, a phenotype opposite of α2δ-1 loss. Loss of TSP1/2 reduced excitatory synapse number significantly in adult brains. However, in the ACC of TSP1/2 knockout mice, there was still training-induced excitatory synaptogenesis, likely through the upregulation of TSP4, a TSP isoform that is also synaptogenic. Unexpectedly, we also found a significant increase in inhibitory synapse number and function in the ACC of TSP1/2 knockout mice, which was eliminated after training. Finally, we found that astrocyte-specific ablation of TSP1/2 in developing but not adult astrocytes is sufficient to reduce performance during high-effort tasks. Taken together, our study highlights the importance of developmental astrocyte-derived synaptogenic cues TSP1 and 2 in establishing excitatory and inhibitory circuits that control effort during operant conditioning in adults.

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Section: Conditional Deletion Of Tsp1/2 In Adult Astrocytes Does Not ...mentioning

confidence: 99%

Section: Conditional Deletion Of Tsp1/2 In Adult Astrocytes Does Not ...mentioning

confidence: 99%

Astrocytic thrombospondins 1 and 2 are required for cortical synapse development controlling instrumental performance

Lawal,

Ulloa Severino,

Wang

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

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GEARBOCS: An Adeno Associated Virus Tool forIn VivoGene Editing in Astrocytes

Bindu

Tan

Savage

et al. 2023

Preprint

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In the mammalian central nervous system (CNS), astrocytes are indispensable for brain development, function, and health. However, non-invasive tools to study astrocyte biology and function in vivo have been limited to genetically modified mice. 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, 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 gene knock-in (Gene-TRAP) strategies. We deployed GEARBOCS to determine whether cortical astrocytes express Vamp2 protein. The presence of Vamp2-positive vesicles in cultured astrocytes is well-established, however, Vamp2 protein expression in astrocytes in vivo has proven difficult to ascertain due to its overwhelming abundance in neurons. Using GEARBOCS, we delineated the in vivo astrocytic Vamp2 expression and found that it is required for maintaining excitatory and inhibitory synapse numbers in the visual cortex. GEARBOCS strategy provides fast and efficient means to study astrocyte biology in vivo.

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GEARBOCS: An Adeno Associated Virus Tool forIn VivoGene Editing in Astrocytes

Cited by 2 publications

References 91 publications

Astrocytic thrombospondins 1 and 2 are required for cortical synapse development controlling instrumental performance

Astrocytic thrombospondins 1 and 2 are required for cortical synapse development controlling instrumental performance

GEARBOCS: An Adeno Associated Virus Tool forIn VivoGene Editing in Astrocytes

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