title: Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines.
abstract: The 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.
authors.affiliation: Department of Pharmacology/Toxicology and Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216, USA.
authors.forename: Tsui-Ting
authors.initials: TT
authors.lastname: Ho
authors.affiliation: Department of Pharmacology/Toxicology and Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216, USA.
authors.forename: Nanjiang
authors.initials: N
authors.lastname: Zhou
authors.affiliation: Department of Pharmacology/Toxicology and Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216, USA.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Pharmacology/Toxicology and Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216, USA.
authors.forename: Pratirodh
authors.initials: P
authors.lastname: Koirala
authors.affiliation: Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001, China.
authors.forename: Min
authors.initials: M
authors.lastname: Xu
authors.affiliation: System Biosciences, Mountain View, CA 94043, USA.
authors.forename: Roland
authors.initials: R
authors.lastname: Fung
authors.affiliation: System Biosciences, Mountain View, CA 94043, USA.
authors.forename: Fangting
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Pharmacology/Toxicology and Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216, USA ymo@umc.edu.
authors.forename: Yin-Yuan
authors.initials: YY
authors.lastname: Mo
mesh_terms: D020137:Base Pair Mismatch; D015153:Blotting, Western; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D022661:RNA, Untranslated / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D035683:MicroRNAs; D022661:RNA, Untranslated