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

Ho, Tsui-Ting; Zhou, Nanjiang; Huang, Jianguo; Koirala, Pratirodh; Xu, Min; Fung, Roland; Wu, Fangting; Mo, Yin‐Yuan

doi:10.1093/nar/gku1198

Cited by 225 publications

(211 citation statements)

References 41 publications

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“…However, it is often necessary to remove the whole sequence of the targeted gene or a large DNA fragment surrounding the promoter or the whole gene (8,11). This can cause a misinterpretation of the function of the targeted gene because it is possible that a gene on the complementary DNA strand overlaps with the targeted gene and/or its regulatory sequence or that a partial coding sequence is also removed due to this overlap (e.g.…”

Section: Discussionmentioning

confidence: 99%

Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes

Liu

Han

Yuan

et al. 2017

Journal of Biological Chemistry

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Edited by Joel GottesfeldThe type II bacterial CRISPR/Cas9 system is a simple, convenient, and powerful tool for targeted gene editing. Here, we describe a CRISPR/Cas9-based approach for inserting a poly(A) transcriptional terminator into both alleles of a targeted gene to silence protein-coding and non-protein-coding genes, which often play key roles in gene regulation but are difficult to silence via insertion or deletion of short DNA fragments. The integration of 225 bp of bovine growth hormone poly(A) signals into either the first intron or the first exon or behind the promoter of target genes caused efficient termination of expression of PPP1R12C, NSUN2 (protein-coding genes), and MALAT1 (nonprotein-coding gene). Both NeoR and PuroR were used as markers in the selection of clonal cell lines with biallelic integration of a poly(A) signal. Genotyping analysis indicated that the cell lines displayed the desired biallelic silencing after a brief selection period. These combined results indicate that this CRISPR/Cas9-based approach offers an easy, convenient, and efficient novel technique for gene silencing in cell lines, especially for those in which gene integration is difficult because of a low efficiency of homology-directed repair.Previous studies indicate that non-protein-coding genes, especially those that encode long non-coding RNAs (lncRNAs), 2 play a key role in gene regulation and are involved in many biological processes, e.g. cell growth, epigenetic regulation, cancer development, and human disease (1-4). Compared with protein-coding genes, silencing long non-protein-coding genes by insertion or deletion of small DNA fragments is difficult as the lncRNA function is primarily subject to conformational changes. Recently, a CRISPR/Cas9-based system was developed as a novel tool for gene editing (5, 6). As a simple, convenient, and efficient system, it has been used for gene editing in a variety of organisms (7). The genes edited with this system include protein coding and non-protein coding genes. Two strategies have been employed for permanently silencing nonprotein-coding genes using large genomic deletions with the CRISPR/Cas9 system. One strategy is to completely delete the given gene using multiple guide RNAs (gRNAs) targeting the 5Ј-and 3Ј-flanking sequences (8 -10). The alternative strategy is to delete core promoter sequences of the given gene (11). The limitation of these strategies is that the deletion of a large genomic fragment may alter the function of the gene of interest due to removal of the potential regulatory elements or other functional genes around the targeted genomic region.This study was designed to silence three genes, including the lncRNA gene, MALAT1, by biallelic integration of a poly(A) signal using the CRISPR/Cas9 system and thus developing an easy, convenient, and efficient approach to silencing gene with the advantages of both the CRISPR/Cas9 and poly(A) signal approaches. First, a poly(A) signal was optionally inserted into three designated sites (immediately behind the...

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Section: Discussionmentioning

confidence: 99%

Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes

Liu

Han

Yuan

et al. 2017

Journal of Biological Chemistry

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“…With the recent rapid advancements in genetic engineering technologies including TALEN and CRISPR-Cas9, it is becoming increasingly straightforward to delete small non-coding RNAs in animal cells. [54][55][56] Moreover, as transgenic animals devoid of specific microRNAs are becoming increasingly accessible from different species including mouse, 57 Drosophila 58 and nematode, 59 we anticipate that TargetLink will gain broad applications for defining the target set of a specific microRNA in different biologic systems including cultured cells and freshly dissected primary tissues. Finally, as discussed earlier, as we continue to optimize the experimental procedures of TargetLink to increase RNA recovery from affinity purification, to adjust processing crosslinked RNA for library preparation and to enhance crosslinking efficiency, we speculate that we ought to be able to further broaden the application of TargetLink by omitting knockout cells as a future enhancement of this method.…”

Section: Discussionmentioning

confidence: 99%

TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells

Chen

et al. 2017

RNA Biology

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MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.

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“…Making use of two gRNAs, the dual CRISPR system is suited for perturbing lncRNAs in contrast to conventional methods, which may lead to insertions or deletions, not enough to eliminate the function of lncRNAs 70, 72 (Table 1). …”

Section: Methods Utilized For Determination Of Lncrna Functionmentioning

confidence: 99%

State of the art technologies to explore long non‐coding RNAs in cancer

Salehi

Taheri

Azarpira

et al. 2017

J Cellular Molecular Medi

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Long non‐coding RNAs (lncRNAs) comprise a vast repertoire of RNAs playing a wide variety of crucial roles in tissue physiology in a cell‐specific manner. Despite being engaged in myriads of regulatory mechanisms, many lncRNAs have still remained to be assigned any functions. A constellation of experimental techniques including single‐molecule RNA in situ hybridization (sm‐RNA FISH), cross‐linking and immunoprecipitation (CLIP), RNA interference (RNAi), Clustered regularly interspaced short palindromic repeats (CRISPR) and so forth has been employed to shed light on lncRNA cellular localization, structure, interaction networks and functions. Here, we review these and other experimental approaches in common use for identification and characterization of lncRNAs, particularly those involved in different types of cancer, with focus on merits and demerits of each technique.

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

Cited by 225 publications

References 41 publications

Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes

Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes

TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells

State of the art technologies to explore long non‐coding RNAs in cancer

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