An AAV-CRISPRCas9 strategy for gene editing across divergent rodent species: Targeting neural oxytocin receptors as a proof of concept

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What is the main point of this article

The main point of this article is to highlight the potential of using comparative gene editing approaches to enhance the translatability of neuroscientific research, with a specific focus on manipulating neural circuits in multiple species. The authors demonstrate the feasibility of a viral-mediated CRISPR/Cas9 strategy to target the oxytocin receptor gene in over 80 rodent species, reducing receptor levels without causing neuronal toxicity. This cross-species gene editing tool is valuable for comparative neuroscience as it eliminates the need for extensive validation in each individual species, contributing to a better understanding of general neural circuit functioning and the role of social behaviors in a species-specific manner .

Next are the main points of each section of the article

Design of viral strategy to target Oxtr in multiple rodents

Researchers have successfully designed a viral vector-mediated tool to reduce OXTR density in the brains of multiple rodent species. They utilized a similar approach to their previous AAV-CRISPR/Cas9 strategy, but this time they used gRNAs targeting conserved Oxtr sequences across rodent species. They identified two conserved regions in the Oxtr coding sequences that were suitable for gRNA targeting, and these regions were found to be located in or just before the second transmembrane domain. This information is depicted in Figure 1A.

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Validation of the viral strategy in the prairie vole

1. AAV-gRNA(ΔOXTR) vectors combined with AAV-Cas9 vectors effectively target the Oxtr coding regions in the nucleus accumbens (NAc) of prairie voles. (Fig. 1B)
2. A T7 endonuclease assay confirms successful AAV-CRISPR/Cas9-mediated genomic editing in the NAc tissue, with specific mutagenesis observed in the Oxtr coding sequence. (Fig. 1C)
3. Autoradiography demonstrates that both AAV-gRNA(ΔOXTR) vectors significantly reduce 125I-OVTA binding in the NAc, indicating disruption of functional OXTR production. (Fig. 1, D and E)
4. The reduction in OXTR levels is not due to neuronal cell death, as both AAV-ΔOXTR-injected and sham-injected hemispheres show similar numbers of NeuN-positive cells. (Fig. 1, D and E)
5. RNAscope in situ hybridization confirms the faithful expression of spCas9 in AAV-injected hemispheres. (Fig. 1G)

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The viral strategy does not afect AVPR1A levels in the prairie vole

1. The gRNA(ΔOXTR) sequences do not align to the prairie vole Avpr1b gene, indicating low potential for off-target effects.
2. The gRNA(ΔOXTR.2) sequence shows the highest sequence homology to the Avpr1a gene, making it the most likely off-target gene in the prairie vole genome for this gRNA.
3. The gRNA(ΔOXTR.1) sequence differs from the prairie vole Avpr1a gene by 8 base pairs.
4. The specificity of the gRNAs was functionally assessed by targeting a region where Avpr1a is expressed (ventral pallidum) and observing AVPR1A autoradiography.
5. The AVPR1A levels in the targeted area were not disrupted, demonstrating the selectivity of the gRNA(ΔOXTR)s.

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Eficacy of the viral strategy in five more rodent species

1. AAV-gRNA(ΔOXTR.1) significantly reduced OXTR (oxytocin receptor) levels across species and brain areas. However, the efficacy was lower in spiny mice compared to other species (fig. S1). This observation is supported by the finding that the target sequence of gRNA(ΔOXTR.1) in spiny mice lacks a PAM sequence, potentially explaining its decreased effectiveness.
2. To address the reduced effectiveness in spiny mice, a second batch of animals was injected with AAV-gRNA(ΔOXTR.2), which was designed with a target sequence located next to a PAM site. This second vector resulted in a strong reduction in OXTR levels.

Therefore, the main conclusion from the section is that the viral strategy, specifically AAV-gRNA(ΔOXTR.1) and AAV-gRNA(ΔOXTR.2), successfully reduced oxytocin receptor levels in multiple rodent species and brain areas.

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The viral strategy targets a wide range of rodent species

1. gRNA(ΔOXTR.1) is predicted to work in 15 out of 30 rodent species based on alignment with RefSeq Oxtr sequences. (Figure 4)
2. gRNA(ΔOXTR.2) is predicted to work in 23 out of 30 rodent species based on alignment with RefSeq Oxtr sequences. (Figure 4)
3. gRNA(ΔOXTR.1) perfectly aligns in 43 out of 231 publicly available rodent genomes, indicating potential functionality. (Figure 4)
4. gRNA(ΔOXTR.2) perfectly aligns in 80 out of 231 publicly available rodent genomes, indicating potential functionality. (Figure 4)
5. Considering the above alignments, these viral vectors are likely to function in at least 81 rodent species. (Table 1)
6. The presence of perfect alignment does not guarantee specific targeting of the Oxtr gene, especially in genomes that are not annotated. Sequence homology analysis with other Oxtr sequences is recommended for confirmation. (Figure 4)
7. The use of the blastn algorithm and NCBI BLAST+ software suite is a valuable tool for identifying potential target species for the viral vectors. (N/A)

The viral strategy targets a wide range of nonrodent mammalian species

1. gRNA(ΔOXTR.1) is predicted to specifically target the Oxtr gene in five nonrodent mammalian species. [Conclusion from Table 2]
2. gRNA(ΔOXTR.2) is predicted to be effective in targeting the Oxtr gene in 67 nonrodent mammalian species. [Conclusion from Table 2]
3. gRNA(ΔOXTR.2) is predicted to target vasopressin 1b (Avpr1b) in 127 nonrodent mammalian species. [Conclusion from Table 3]

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