shRNAmir and CRISPR sgRNA Library Construction Core

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PROJECT SUMMARY / ABSTRACT Core B (Pipkin) Short hairpin RNAs in microRNA contexts (shRNAmirs) and CRISPR-Cas9 guide RNA (sgRNA) are powerful tools for experimental RNA interference (RNAi) and gene-disruption in the immune system, respectively. However, virtually all current approaches require pre-activation of naive cells to render them susceptible for gene transfer, typically with retroviral or lentiviral vectors, and cause constitutive RNAi or immediate gene-disruption. These issues complicate studying genes that function prior to retroviral delivery, and clarifying the temporal requirements of genes at different stages of immune responses. In Core B, we describe development of multiple innovative tools and methods that substantially expands the utility of shRNAmirs and sgRNAs to study gene function. We demonstrate generation of unmanipulated naive CD4 and CD8 T cells carrying conditionally regulated retroviral constructs that enable conducting inducible and reversible RNAi in vivo, and extend this system to a novel approach that facilitates large in vivo pooled screens using inducible shRNAmirs in untouched naive cells. We also demonstrate CRISPR-Cas9 guide RNA (sgRNA) gene disruption in primary B and T cells, and its use during in vivo immune responses to analyze B and T cell function. The combination of these shRNAmir and sgRNA approaches are synergistic and constitute an innovative and rigorous experimental platform for rapidly dissecting gene function in T and B cells during in vivo immune responses. Core B builds on multiple existing libraries of high-fidelity shRNAmir clones, which include those that target all genes encoding mammalian chromatin regulator factors (312 genes, ~1,700 shRNAmirs), and all mammalian transcription factors (2,083 genes, ~8,000 shRNAmirs). The overarching goal of Core B is to provide essential tools for executing the proposed in vivo loss-of-function studies proposed in Projects 1-3 of this P01. Our specific objectives are to sub- clone pooled shRNAmir libraries using an inducible vector for conditional pooled screens in naive T cells that are proposed in Projects 2 and 3 (Aim 1); to produce high-quality, ready-to-transfect retroviral plasmid DNA from cloned libraries of shRNAmirs and sgRNAs for Projects 1-3 (Aim 2); and to re-apply existing arrayed shRNAmir libraries and build custom shRNAmir and gRNA clone sets in appropriate vectors based on gene targets prioritized during the evolution of Projects 1-3 to facilitate detailed follow-up studies (Aim 3).