Abstract 2950: CRISPR/saCas9 and CRISPR/spCas9 systems for combinatorial genetic screens (CRISPR-KO, CRISPRa, CRISPRi)

Abstract

Abstract This study explores the utilization of orthogonal CRISPR-based gene editing/modulation systems for combinatorial genetic screens using CRISPR knockout (CRISPR-KO), CRISPR activation (CRISPRa), and CRISPR interference (CRISPRi) functionalities. S. aureus (sa)Cas9 is an alternative nuclease to S. pyogenes (sp)Cas9 in scenarios where the latter cannot be used, or when multiple independent CRISPR systems need to be simultaneously expressed in the same cell. In this study we set out to explore the feasibility of utilizing different combinations of saCas9 and spCas9 CRISPR systems to achieve the simultaneous inactivation (via CRISPR-KO or CRISPRi) and transactivation (via CRISPRa) of different target genes in the same host cell. For this purpose, a complete set of tools for CRISPR/saCas9 gene editing and gene modulation was developed, compatible with CRISPR/spCas9 co-expression. Specifically, we developed and validated optimized saCas9 and sg(sa)RNA lentiviral and AAV vectors, dual expression (sp)/(sa)sgRNA lentiviral library vectors, as well as (sa)CRISPR-KO, (sa)CRISPRi, (sa)CRISPRa fluorescence-based activity kits for the functional validation of saCas9 expressing cell lines. Results demonstrating the feasibility of the orthogonal screens will be presented, with different combinations of CRISPR-KO, CRISPRa, and CRISPRi systems in multiple cell lines. Citation Format: Nadya Isachenko, Gayane Aleksanyan, Paul Diehl, Donato Tedesco. CRISPR/saCas9 and CRISPR/spCas9 systems for combinatorial genetic screens (CRISPR-KO, CRISPRa, CRISPRi) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2950.