Campbell Rianne R, Green Mikah, Choi Eric Y, Wulff Andreas B, Siclair Allison N, Khatri Smirti, Virata Geralin, Barrett Christina, Key Symphanie, Patel Samir, Rowell Mary Beth, Franco Daniela, Ganapathy-Kanniappan Shanmugasundaram, Mathur Brian N, Lobo Mary Kay
Department of Neurobiology University of Maryland, School of Medicine, Baltimore, Baltimore, MD USA.
Department of Pharmacology and Physiology, University of Maryland School of Medicine, HSF III 9179, Baltimore, MD 21201, USA.
bioRxiv. 2025 Jun 27:2025.06.26.661721. doi: 10.1101/2025.06.26.661721.
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.
纹状体中的两种主要细胞类型，即多巴胺受体1和腺苷受体2a棘状投射神经元（D1-SPNs和A2A-SPNs），在调节运动和奖赏相关行为中具有不同作用。Cre选择性CRISPR-dCas9系统可通过基因特异性单向导RNA（sgRNAs）对基因表达进行细胞类型特异性的、基于表观基因组的操控，并有潜力阐明纹状体亚型介导行为背后的分子机制。最近构建了条件转基因Rosa26:LSL-dCas9-p300小鼠，以实现dCas9-p300在Cre驱动的细胞类型特异性下的强劲表达。该系统利用p300，一种组蛋白乙酰转移酶，它通过解开染色质并使基因组的该区域更易于转录来调节基因表达。将Rosa26-LSL-dCas9-p300小鼠与Drd1-Cre和Ador2a-Cre小鼠配对，以生成Drd1-Cre:dCas9-p300和Ador2a-Cre:dCas9-p300小鼠品系，并在不存在sgRNAs时进行行为表型分析。Drd1-Cre:dCas9-p300和Ador2a-Cre:dCas9-p300均具有spCas9 mRNA的细胞类型特异性表达。基线行为评估显示，在不存在sgRNA的非靶向状态下，与所有基因型同窝小鼠相比，Drd1-Cre:dCas9-p300小鼠表现出重复旋转行为、运动亢进和奖赏学习获得增强。相比之下，Ador2a-Cre:dCas9-p300与其同窝小鼠相比在行为上未表现出任何变化。对背侧纹状体D1-SPNs的电生理记录显示，Drd1-Cre:dCas9-p300小鼠的输入电阻增加，自发兴奋性突触后电流幅度增加，共同表明D1-SPNs的兴奋性驱动增强。总体而言，这些数据证明了在研究中验证CRISPR-dCas9品系的必要性。此外，Drd1-Cre:dCas9-p300品系有潜力用于研究刻板行为和奖赏学习的潜在机制。
