Core C - Genomics, CRISPR, and Gene Perturbation Core

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

Investigators

Linked publications, trials & patents

Abstract

The overall goal of this IR-PPG is to use cellular and transcriptomic guided approaches to define the causal mechanisms by which PD1 and LAG3 control diverse types of T cells in the context of autoimmunity, cancer, and chronic infection. A major approach to be used throughout this Program is single cell sequencing, including single cell RNA sequencing (scRNA-seq) and/or scATAC-seq linked to downstream CRISPR screening to define causal mechanisms. Thus, the purpose of the Genomics, CRISPR, and Gene Perturbation Core (Core C) is to provide essential and centralized integrated analysis of key datasets and design of common CRISPR screening libraries for use across all three Projects in this Program. In addition, this Core will provide advanced computational analysis and provide a platform for a retroviral or lentiviral (RV/LV)-enforced expression and knockdown to use to directly test in vivo individual genes and pathways identified from CRISPR screening. Thus, Core C will provide integrated bioinformatic and computational analytical platforms and data integration services coupled to downstream RV/LV-enforced expression and knockdown as well as in vivo CRISPR/Cas9-focused genetic screening. The Aims are: AIM 1: To provide advanced computational support and identify mechanistic targets of PD1 and/or LAG3 for CRISPR-based screening through cross-Project data integration, advanced computational analyses, and sgRNA library design. Core C will provide five services: [i] support data ingest and analysis for cross- Project analysis and CRISPR library development; [ii] ingest, quality control, and normalize external datasets for integrated analysis with IR-PPG datasets; [iii] perform integrated analysis to identify common and disease- specific biology linked to PD1/LAG3; [iv] assist the Projects in implementing novel analytical pipelines and data analysis tools; and [v] develop focused CRISPR libraries. AIM 2: To enable in vivo CRISPR/Cas9 screening and provide a retrovirus/lentivirus-enforced expression and knockdown platform for in vivo testing of genes of interest (GOI) regulated by PD1 and LAG3. Several robust in vivo CRISPR/Cas9 screening platforms for genetic perturbation in T cells in mouse models of disease have been established in the IR-PPG to test causality of genes identified through transcriptional analysis. Core C also has developed a robust retrovirus/lentivirus gene manipulation platform to advance from causal implications to mechanistic understanding of GOI. Core C will provide tools to test causality and interrogate mechanisms through two services: [i] provide common CRISPR libraries and facilitate CRISPR library construction in retrovirus/lentivirus for in vivo screens; and [ii] provide retrovirus/lentivirus-based tools to test mechanisms of GOI implicated downstream of PD1 and/or LAG3. IR-PPG Interactions: Core C will analyze samples and integrate data for the three Projects. Core C will interact heavily with Cores A, B, and D to identify gene targets for novel mouse strains and antibody development.