Abstract Body:
Introduction:
Pathological stimuli trigger diverse cardiomyocyte stress responses that must be tightly regulated to promote adaptive remodeling while limiting maladaptive outcomes. However, the transcriptional regulators governing stress-responsive gene programs have not been fully identified, in part due to the high cost and limited throughput of traditional in vivo genetic approaches.
Hypothesis:
We hypothesize that the development and use of a novel reporter assay for massively parallel assessment of CRISPR-based somatic mutations will enable cost-effective high-throughput identification of regulators of cardiomyocyte stress responses in vivo.
Methods:
We have developed AAV/CRISPR-based massively parallel perturbation reporter assays (MPPRAs) to perform high-throughput in vivo forward genetic screens for regulators of Nppa expression and select stress-responsive synthetic cis-regulatory elements in neonatal mouse cardiomyocytes. Compared with conventional pooled CRISPR screens and approaches incorporating single-cell RNA sequencing (e.g., Perturb-seq), this strategy substantially improves cost efficiency, scalability, and technical simplicity.
Conclusions:
Using this technique, we identified numerous candidate regulators that modulate cardiomyocyte stress-response pathways, with chromatin modifiers prominently represented among the top hits. Notably, BAF45c (Dpf3) and BAF47 (Smarcb1), components of the BAF chromatin remodeling complex, emerged as common regulators across multiple screens. Our preliminary analyses of Dpf3 and Smarcb1 confirm that loss-
of-function results in altered gene regulatory network activity. In conclusion, this work establishes AAV/CRISPR-based MPPRAs as a scalable in vivo platform for high-throughput functional genetic screening in the heart, overcoming key limitations of traditional models. These findings uncover previously unrecognized regulators of cardiomyocyte stress responses and provide a framework for systematically dissecting cardiac gene regulatory networks, with potential to accelerate the discovery of new therapeutic targets for heart disease.