GEARBOCS: An Adeno Associated Virus Tool for In Vivo Gene Editing in Astrocytes

Reviewer #1 (Public review):

Summary:

The manuscript by Bindu et al. created an AAV-based tool (GEARAOCS) to perform in vivo genome editing of mouse astrocytes. The authors engineered a versatile AAV vector that allows for gene deletion through NHNJ, site-specific knock-in by HDR, and gene trap. By utilizing this tool, the authors deleted Sparcl1 virally in subsets of astrocytes and showed that thalamocortical synapses in cortical layer IV are indeed reduced during a critical period of ocular dominance plasticity and in adulthood, whereas there is no change in excitatory synapse number in cortical layer II/III. Furthermore, the authors made a VAMP2 gene-trap AAV vector and showed that astrocyte-derived VAMP2 is required for the maintenance of both excitatory and inhibitory synapses.

Strengths:

This AAV-based tool is versatile for astrocytic gene manipulation in vivo. The work is innovative and exciting, given the paucity of tools available to probe astrocytes in vivo.

Weaknesses:

Several important considerations need to be made for the validation and usage of this tool, including:

Major points:

(1) Efficiency and specificity of spCas9-sgRNA mediated gene knockout in astrocytes. In Figure 3, the authors utilized Sparcl1 gene deletion as the proof-of-principle experiment. The readout for Sparcl1 KO efficiency is solely the immunoreactivity using an antibody raised against Sparcl1. As the method is based on NHEJ, the indels can be diverse and can occur in one allele or two. For the tool and proof-of-principle experiment, it will be important to know the percentage of editing near the PAM site, as well as the actual sequences of indels. This can be done by single-cell PCR of edited astrocytes, similar to the published work (Ye... Chen, Nature Biotechnology 2019).

(2) Along the same line, the authors showed that GEARBOCS TagIn of Sparcl1 resulted in 12.49% efficiency based on the immunohistochemistry of mCherry tag. It is understandable that the knock-in efficiency is much reduced as compared to gene knockout. However, it remains unclear if those 12.49% knock-in cells represent sequence-correct ones, as spCas9-mediated HDR is also an error-prone process, and it may accidentally alter nucleotides near the PAM site without causing the frameshift. The author will need to consider the related evidence or make comments in the discussion.

(3) What are the efficiencies of Sparcl1 GEARBOCS GeneTrap (Figure 3V) and Vamp2 GeneTrap and HA TagIn (Figure 5)?

Minor points:

(1) Figure 3H-J. The authors only showed the representative images of Sparcl1 KO. Please consider including the control (without gRNA), given that there are still many Sparcl1+ signals in Figure 3I (likely because of its expression in other cell types?).

(2) In figure 3Q-T, it appears that some Cas9-EGFP+ astrocytes (Q) do not express Sparcl1 (R). Is Sparcl1 expressed in subsets of astrocytes? Does Cas9-EGFP or Sparcl1-TagIn alter Sparcl1 endogenous expression?

(3) On Page 8, for the explanation of the design of the GEARBOCS construct, the authors have made a self-citation (#43). That was a BioRxiv paper that is being reviewed currently.

(4) For Figures 4 and 6, the graphs seem to be made in R with the x-axis labeled as "Condition". The y-axis labels are too small to read properly, especially in print. It would be better to make the graphs clearer like Figure 2 and Figure 3.

(5) On Page 13, "Figures 3V-Y" were referred to. However, there are no Figures 3W, X, and Y.

(6) There are a few typos in the manuscript, including line 900 "immunofluorescence microscopy images of a Cas9-EGFP-positive astrocytes (green)".

Reviewer #2 (Public review):

Summary:

The present study described GEARBOCS, an adeno-associated virus tool for in vivo gene editing in astrocytes. This tool is timely and important for glial biologists who often are troubled by efficient gene targeting in astrocytes. Overall the significance of the finding is valuable, and the strength of the evidence is solid. Presumably, there will be great potential associated with GEARBOCS applications in the future.

Strengths:

As efficient tools for targeting non-neuronal cells in the brains are rather limited for astrocytes and microglia, GEARBOCS adds to the small pool of currently available tools and will provide new options for glial biologists studying these tools. As the study revealed, GEARBOCS are capable of knockout and knockin manipulations for genes of interest, also ascribed with reporter tracking and gene-trap strategy. The promising multi-functional tool will advance our understanding of astrocytes and help to further elucidate the mechanism of neuron-glia interaction.

Weaknesses:

Even though the tool seems promising and powerful. the authors failed to provide more evidence on the robustness and specificity of GEARBOCS. Also, the advantages of GEARBOCS over some of the traditional methods were not clearly stated. Some of these concerns are described below.

Reviewer #3 (Public review):

Summary:

Sivadasan Bindu et al. developed a CRISPR/Cas9-based gene-editing strategy using a single AAV vector, named GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/Cas9 System), which enables precise genome manipulation in astrocytes. This tool was shown to effectively perform knockout, tagging, and reporter knock-in gene modifications. The utility of GEARBOCS was demonstrated in two cases: establishing astrocytes as essential for the synaptogenic factor Sparcl1 in thalamocortical synapse maintenance, and revealing that cortical astrocytes express the Vamp2 protein, which is vital for maintaining synapse numbers.

Strengths:

Astrocytes play a crucial role in brain development and function, but studying them in vivo has been challenging due to limited molecular tools for manipulation. Sivadasan Bindu et al. developed a valuable system called GEARBOCS for effective astrocyte infection via retro-orbital injection.

Weaknesses:

The manuscript provides data only from the cerebral cortex and results from P42. Additional data from other brain regions and various time points (e.g., P0-15) are needed. Results from local injection experiments would also enhance the utility of this tool for the broader glial research community.