Scientific Core: CRISPRi mouse husbandry and AAV delivery

Stanford University, Stanford CA

Investigators

Abstract

PROJECT SUMMARY: CRISPR Mouse Husbandry and AAV Delivery (Mouse Core) The overarching goal of this Scientific Core is to support in vivo Perturb-seq experiments to map gene programs in a consistent, coordinated fashion across all three Projects by providing standardized mouse models and adeno-associated virus (AAV) guide RNA (gRNA) libraries. All three Projects will use the same Cre-inducible dCas9-KRAB (CRISPRi) mouse line with the same gRNA library packaged into AAVs for in vivo Perturb-seq. To investigate candidate causal gene function in an appropriate vascular disease model, each Project will require breeding the CRISPRi allele with other disease-specific alleles and lineage-tracing marker alleles. Mouse alleles employed in these studies are being sourced from multiple laboratories in different backgrounds. Thus, it is critical that the individual mouse lines be validated, and their authenticity be managed in accordance with strict lineage control through genotyping all alleles and maintaining comprehensive breeding records. Centralized mouse model generation in a homogenous relevant background is key to comparing Perturb-seq data for the same genes across different cell types and organ systems, and to mapping gene programs in a consistent, coordinated fashion across all 3 Projects. To this end, we are proposing four Specific Aims: 1) to establish mouse colonies that will provide Cre-inducible vascular cell-specific conditional knockout and lineage tracing for the in vivo studies to validate coronary artery disease (CAD) and pulmonary arterial hypertension (PAH) genes in vascular disease models; 2) to combine the Cre-inducible dCas9-KRAB (CRISPRi) allele with CAD and PAH animal disease models; 3) to generate high quality AAVs with pooled gRNA libraries that efficiently transduce endothelial cells (ECs) in the aorta, brain, and lung; and 4) to optimize and improve technologies required for in vivo Perturb-seq. Through these activities, the Mouse Core will provide breeder mice of the needed genotype for expansion to each Project with breeding and genotyping instructions. The Core will also provide high-titer, high-purity AAVs containing gRNA libraries for in vivo Perturb-seq along with dosing information. Working closely with personnel from each of the 3 Projects, the Mouse Core will help improve the efficiency of in vivo Perturb-seq throughout the 5-year program. This Core will be administered by Thomas Quertermous (Overall PI and Core PI), Chong Park (Overall Project Manager, and Co-I on this Core), and seasoned senior research assistant Ramendra Kundu. Drs. Quertermous and Kundu have worked together for two decades generating and phenotyping genetically modified mouse models, and Dr. Park was previously the Project Manager for the Keck miRNA Knockout Mouse Project and Director of the Embryonic Stem Cell Targeting Core at UCSF. The Mouse Core will bring together an unparalleled set of technologies in the cardiovascular field to study two of the leading causes of human disability and death, and support collaborations on understanding shared genetics between these diseases and common cell types.