CRISPitope: A generic platform to model target antigens for adoptive T cell transfer therapy in mouse tumor models

Abstract

Summary This protocol details the procedure for CRISPR-assisted insertion of epitopes (CRISPitope), a flexible approach for generating tumor cells expressing model CD8 + T cell epitopes fused to endogenously encoded gene products of choice. CRISPitope-engineered tumor cells can be recognized by T cell receptor-transgenic (TCRtg) CD8 + T cells that are widely used in immunology research. Using mice inoculated with CRISPitope-engineered tumor cells, researchers can investigate how the choice of the target antigen for T cell immunotherapies influences treatment efficacy and resistance mechanisms. For complete details on the use and execution of this protocol, please refer to Effern et al. (2020) .

Preclinical evidence for the use of anti‐Trop‐2 antibody‐drug conjugate Sacituzumab govitecan in cerebral metastasized castration‐resistant prostate cancer

“…pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138; http://n2t.net/addgene:48138; RRID: Addgene_48138). Generation and cloning of Trop‐2 single‐guide RNA (sgRNA) CRISPR‐Cas9 KO plasmids was performed following the established STAR protocol 16 . PX458, was digested with the restriction enzyme BbsI (#R0539L, New England Biolabs).…”

“…Generation and cloning of Trop‐2 single‐guide RNA (sgRNA) CRISPR‐Cas9 KO plasmids was performed following the established STAR protocol. 16 PX458, was digested with the restriction enzyme BbsI (#R0539L, New England Biolabs). Two different sgRNA sequences targeting the Trop‐2 gene were cloned into PX458.…”

Membranous NECTIN-4 Expression Frequently Decreases during Metastatic Spread of Urothelial Carcinoma and Is Associated with Enfortumab Vedotin Resistance

“…13). The generation and cloning of the NECTIN-4 single-guide RNA (sgRNA) CRISPR-Cas9 KO plasmids was performed following our established STAR protocol (14). PX458 was digested with the restriction enzyme BbsI (New England Biolabs, catalog no: R0539L).…”

“…NECTIN-4_KO1_TS: caccgTGGGACACCACACAAGTCAG NECTIN-4_KO1_BS: aaacCTGACTTGTGTGGTGTCCCAc NECTIN-4_KO2_TS: caccgCTCCCTCATACAACTGGACA NECTIN-4_KO2_BS: aaacTGTCCAGTTGTATGAGGGAGc Establishment of polyclonal NECTIN-4 KO cultures HT1376 cells were transfected with the NECTIN-4 KO plasmids using FuGENE Transfection Reagent (Promega, catalog no: E2311) with a FuGENE to DNA Ratio of 4:1 (14). Seventy-two hours after transfection, the 15% strongest GFP-positive cells were sorted via the Flow Cytometry Core Facility (FCCF; https://btc.uni-bonn.de/fccf/) of the UKB using the FACS Aria III (BD Biosciences) to obtain polyclonal HT1376 NECTIN-4 KO cultures.…”