Abstract 167: Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines

Abstract

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system (CRISPR/Cas) has been recently shown to be a powerful genome-editing tool in a variety of organisms. However, these studies are mainly focused on protein-coding genes. The present study aims to determine whether this technology can be applied to non-coding genes. One of the challenges for knockout of non-coding genes is that a small deletion or insertion generated by the standard CRISPR/Cas system may not necessarily lead to functional loss of a given non-coding gene because of lacking an open reading frame, especially in polyploidy human cell lines. To overcome this challenge, we adopt a selection system that allows for marker genes to integrate into the genome through homologous recombination (HR). Moreover, we construct a dual guide RNA vector that can make two cuts simultaneously at designated sites such that a large fragment can be deleted. With these approaches, we are able to successfully generate knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 in various human cell lines. Finally, we show that the HR-mediated targeting efficiency can be further improved by suppression of the non-homologous end joining pathway. Together, these results demonstrate the feasibility of knockout for non-coding genes by the CRISPR/Cas system in human cell lines. Citation Format: Tsui-Ting Ho, Nanjiang Zhou, Jianguo Huang, Pratirodh Koirala, Min Xu, Roland Fung, Fangting Wu, Yin-Yuan Mo. Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 167. doi:10.1158/1538-7445.AM2015-167