Dopamine receptor 1 specific CRISPRa mice exhibit disrupted behaviors and striatal baseline cellular activity

Campbell, R. R.; Green, M.; Choi, E. Y.; Wulff, A. B.; Siclair, A. N.; Khatri, S.; Virata, G.; Barrett, C.; Key, S.; Patel, S.; Rowell, M. B.; Franco, D.; Ganapathy-Kanniappan, S.; Mathur, B. N.; Lobo, M. K.

doi:10.1101/2025.06.26.661721

bioRxiv · 10.1101/2025.06.26.661721

Dopamine receptor 1 specific CRISPRa mice exhibit disrupted behaviors and striatal baseline cellular activity

Campbell, R. R.·Green, M.·Choi, E. Y.·Wulff, A. B.·Siclair, A. N.·Khatri, S.·Virata, G.·Barrett, C.·Key, S.·Patel, S.·Rowell, M. B.·Franco, D.·Ganapathy-Kanniappan, S.·Mathur, B. N.·Lobo, M. K.

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Abstract

The two main cell types in the striatum, dopamine receptor 1 and adenosine receptor 2a spiny projection neurons (D1-SPNs and A2A-SPNs), have distinct roles in regulating motor- and reward-related behaviors. Cre-selective CRISPR-dCas9 systems allow for cell-type specific, epigenomic-based manipulation of gene expression with gene-specific single guide RNAs (sgRNAs) and have potential to elucidate molecular mechanisms underlying striatal subtype mediated behaviors. Conditional transgenic Rosa26:LSL-dCas9-p300 mice were recently generated to allow for robust expression of dCas9-p300 expression with Cre-driven cell-type specificity. This system utilizes p300, a histone acetyltransferase which regulates gene expression by unwinding chromatin and making that region of the genome more accessible for transcription. Rosa26-LSL-dCas9-p300 mice were paired with Drd1-Cre and Ador2a-Cre mice to generate Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 mouse lines and underwent behavioral phenotyping when sgRNAs were not present. Both Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 have cell-type specific expression of spCas9 mRNA. Baseline behavioral assessments revealed that, under a sgRNA absent nontargeted state, Drd1-Cre:dCas9-p300 mice display repetitive spinning behavior, hyperlocomotion and enhanced acquisition of reward learning in comparison to all genotypic littermates. In contrast, Ador2a-Cre:dCas9-p300 do not exhibit any changes in behavior in comparison to their littermates. Electrophysiological recordings of dorsal striatum D1-SPNs revealed that Drd1-Cre:dCas9-p300 mice have increased input resistance and increased spontaneous excitatory postsynaptic current amplitude, together suggesting greater excitatory drive of D1-SPNs. Overall, these data demonstrate the necessity to validate CRISPR-dCas9 lines for research investigations. Additionally, the Drd1-Cre:dCas9-p300 line has the potential to be used to study underlying mechanisms of stereotypy and reward-learning. Significance StatementUsing CRISPR-based tools to identify cell-type specific epigenomic and transcriptional mechanisms in disease and behavior has high utility for the neuroscience field. Previous limitations related to implementation of CRISPR-editing systems in mice were thought to be overcome by the generation of transgenic mouse lines, including a novel Cre-dependent dCas9-p300 mouse line. Our data shows however that Drd1-Cre:dCas9-p300 mice, generated from breeding Drd1-Cre mice with the dCas9-p300 mice, have cellular and behavioral disruptions under a nontargeted sgRNA absent state. Overall, these data suggest caution in employing CRISPR-dCas9 systems, particularly transgenic mouse lines, for research investigations.

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Campbell, R. R., Green, M., Choi, E. Y., Wulff, A. B., Siclair, A. N., Khatri, S., Virata, G., Barrett, C., Key, S., Patel, S., Rowell, M. B., Franco, D., Ganapathy-Kanniappan, S., Mathur, B. N., Lobo, M. K.. 2025-06-27. Dopamine receptor 1 specific CRISPRa mice exhibit disrupted behaviors and striatal baseline cellular activity. https://doi.org/10.1101/2025.06.26.661721

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