In vivo targeted CRISPR based loss-of-function mutations during neonatal mouse heart development 
Creators: name: Qian, Yunzhe
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2022

description: Technologies that aim to alter DNA advances ever since the discovery of DNA double helix. Previous studies showed that Clustered Regularly Interspaced Short Palindromic Repeats pathway (CRISPR) can edit mammalian genomes in a cost-effective and user-friendly method1. Together with sgRNA (single-guide RNA), CRISPR/Cas9 can be effectively used in vivo to generate controlled mutations with specific cell targets2-3. By simply altering the sequence of sgRNA, we can reprogram Cas9 to target different genomic sites2-3,4. To achieve cardiomyocyte-specific knockout (KO) of the target genes with CRISPR/Cas9, we adopted CRISPR/Cas9 into AAV vector to create recombinant AAV serotype 9 (AAV9) to deliver single guide RNA (sgRNA) into the cardiomyocytes (CM), named CASAAV5. This project is a part of the on-going project in Qian lab. In this project, we will use in vivo perturb-seq to study CM maturation. Briefly, CASAAV will be used in a pooled approach to introduce mutations in each of the selected risk genes and interrogate each gene’s KO effect with single cell RNA-seq (scRNA-seq). We will knock out the risk genes identified from previous Genome-wide Association Studies (GWAS) of congenital and hypertrophic heart disease during neonatal heart maturation and study their function on CM maturation6. As the first in vivo Perturb-Seq on mouse heart development, the project could significantly deepen our understanding of CM maturation. Moreover, compared to classic approach to study gene’s function, in vivo Perturb-Seq can identify cardiac specific effects of genetic perturbations in a high-throughput manner7. 
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