Abstract 5210: Ablation of an immunogenic human endogenous retrovirus in renal cell carcinoma cells through dual-guide CRISPR-Cas9 genome editing
Authors: Chen Long , Cherkasova Elena , Pierre Stephanie , England Savannah , Igboko Muna , Clara Joseph , Barisic Stefan , Parrizzi Angie , Nadal Rosa Rios , Allan David , Chakraborty Mala , Reger Robert , Childs Richard 
Published: April 2023

Abstract
Human endogenous retroviruses (HERVs) comprise 8% of the human genome and can be abnormally expressed in different tumor cells. Our lab discovered a unique and highly immunogenic HERV-E, CT-RCC HERV-E, that is selectively expressed in most clear cell renal cell carcinoma (ccRCC) cells. Although the function of select HERVs in some tumors has been defined, the role of CT-RCC HERV-E in ccRCC remains unclear. To characterize the impact of HERV-E expression on tumor oncogenesis in ccRCC, we developed a highly efficient strategy to ablate CT-RCC HERV-E through dual-guide CRISPR-Cas9. For isolation of edited cells, we knocked in a truncated CD19 (tCD19) into the same gene edited locus using adeno-associated virus (AAV) as donor templates. In order to ablate the entire 8.8 kb HERV-E region, we designed three single guide RNAs (sgRNAs) flanking the CT-RCC HERV-E genomic locus: one at the upstream site, sgRNA HERV-E Upst(1), and two downstream, sgRNA HERV-E Dwst(3) and sgRNA HERV-E Dwst(4). A single sgRNA targeting β2M was used as a negative control. HERV-E knockout was verified via real-time PCR, and the combination of sgRNAs Upst1-Dwst3 were chosen as the guides for all experiments, due to their higher knockout efficiency. We tested the MOIs of 2, 4, and 10 × 105 for AAV transduction following CRISPR knockout. Expression of CD19 was the highest at the MOI of 10 × 105, which was used for all experiments. Two RCC cell lines, RCC-TIU and RCC-UOK220, were used to validate this methodology. Edited ccRCC cells were enriched by CD19 magnetic microbeads, resulting in CD19 expression of over 96%. Genomic DNAs isolated from the two RCC cell lines confirmed simultaneous knockout of HERV-E and knock-in of tCD19. To isolate a population of cells that had homogenous HERV-E knockout, we single-cell sorted and then expanded CD19+ ccRCC cells in vitro. 252 and 145 clones were harvested from RCC-TIU and RCC-UOK220 respectively. PCR analysis of genomic DNA from 48 selected clones of each of the aforementioned cell lines unveiled both monoallelic and biallelic knockout of HERV-E. Eight biallelic knockout clones were harvested and further expanded, which gave rise to four clones from RCC-TIU and three clones from RCC-UOK220. Additional PCR screening confirmed that RCC-TIU A7, A9, and RCC-UOK220 D3 were biallelic knockout clones. Finally, we used digital PCR to examine the expression of two CT-RCC HERV-E transcripts, Env and RCC-8; No expression of these transcripts was found in the aforementioned clones. In summary, we established a highly efficient method to reliably ablate a human endogenous retrovirus in human tumor cells. The dual-guide CRISPR-Cas9 genome editing method utilized here allowed for the successful isolation of tumor cells that had a biallelic knockout of the CT-RCC HERV-E. These knockout cell lines will be utilized to explore the oncogenic impact of CT-RCC HERV-E expression in ccRCC cells.
Citation Format: Long Chen, Elena Cherkasova, Stephanie Pierre, Savannah England, Muna Igboko, Joseph Clara, Stefan Barisic, Angie Parrizzi, Rosa Rios Nadal, David Allan, Mala Chakraborty, Robert Reger, Richard Childs. Ablation of an immunogenic human endogenous retrovirus in renal cell carcinoma cells through dual-guide CRISPR-Cas9 genome editing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5210.