Date Published: March 16, 2017
Publisher: Public Library of Science
Author(s): Krishna Das, David Eisel, Clarissa Lenkl, Ashish Goyal, Sven Diederichs, Elke Dickes, Wolfram Osen, Stefan B. Eichmüller, Hodaka Fujii.
In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.
Targeted genome editing, i. e. introduction of artificial sequence changes at targeted sites within the genome, has become a standard procedure in molecular biology. Recently, the bacterial “Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated nuclease 9” (CRISPR/Cas9) system has evolved as a powerful platform for targeted genome editing. Originally, the CRISPR/Cas9 system represented a prokaryotic adaptive immune defense mechanism protecting bacteria from invasion by foreign genomes, based on the integration of foreign DNA sequences as so called protospacers into the host chromosome, separated by endogenous CRISPR sequences [1, 2]. Meanwhile, the CRISPR/Cas9 system has been adopted for routine targeted genome editing based on induction of sequence-specific DNA double strand breaks (DSB) . In fact, this technique has been successfully applied to genetically modify bacteria  or mammalian cell lines  and was furthermore used for generation of various knockout mouse strains .
Seeking straight forward strategies for the generation of murine MHC deficient tumor cell lines we focused on the CRISPR/Cas9 based technology, as this technique was suggested to outperform classical strategies of targeted genome editing based on ZFPs or TALENs in frequency and precision . In fact, usage of the construct PX458_mβ2m guide #1 was highly efficient, resulting in approximately 80% MHC class I negative B16F10 cells and EO-NY cells, respectively upon transfection, thereby exceeding mutation frequencies described for CRISPR/Cas9 induced genome editing in other murine cell lines .