Abstract Body: Introduction: Single-cell CRISPR screens link genetic perturbations to transcriptional phenotypes, enabling parallel investigation of gene function. Platforms such as Perturb-seq and CROP-seq established these strategies in vitro, and recent studies show AAV delivery can extend CRISPR perturbations to complex tissues in vivo. Yet, these approaches have not been applied to endogenous cardiomyocytes (CMs), highlighting the need for CM-specific systems compatible with standard single cell RNA-seq workflows. Here, we develop a CM-specific in vivo CRISPR platform by integrating CROP-seq polyadenylated gRNA detection with AAV9 delivery.
Methods: We designed an AAV9 vector containing a cTnT-Cre-U6-gRNA-polyA cassette for CM-specific Cre expression and polyadenylated gRNA transcription. The vector was packaged into AAV9 with a validated gRNA targeting Junctophilin-2 (Jph2) and injected subcuaneously into neonatal (P0) Rosa26-LSL-Cas9 mice. These mice harbor Cre-dependent Cas9 and EGFP expression, allowing for tracking of transduced CMs. At P14, CMs were isolated, and the percentage of EGFP-positive cells were quantified using a large particle biosorter. Genomic editing was assessed by PCR amplification of the Jph2 locus followed by Sanger sequencing and Tracking of Indels by Decomposition (TIDE) analysis. Jph2 knockout was validated at transcript and protein levels by qPCR and immunostaining.
Results: Biosorter analysis revealed ~15% and ~55% EGFP-positive CMs following low-dose (5x10⁹ genome copies [GC]/g) and high-dose (5x10¹⁰ GC/g) injections, respectively, confirming CM-specific Cre activation and Cas9 expression. TIDE analysis demonstrated indel formation at the Jph2 target site. Injected mice exhibited reduced Jph2 mRNA levels, and immunostaining confirmed loss of Jph2 protein in EGFP-positive CMs.
Conclusions: These results establish a CM-specific in vivo CRISPR knockout platform compatible with standard single-cell RNA-sequencing workflows. AAV9 enables efficient cardiac delivery, while polyadenylated gRNAs allow direct capture during library preparation without separate barcode constructs or custom sequencing. This approach provides a scalable foundation for pooled perturbation screens in the heart.