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Gene Silencing Using Polya Signal Via CRISPR/Cas9 System Biallelic

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Gene Silencing Using Polya Signal Via CRISPR/Cas9 System Biallelic JBC Papers in Press. Published on February 14, 2017 as Manuscript M116.769034 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.769034 gene silencing using PolyA signal via CRISPR/Cas9 system Biallelic Insertion of a Transcriptional Terminator via CRISPR/Cas9 Efficiently Silences Expression of Protein-coding and Non-coding RNA Genes Yangyang Liu1,2, Xiao Han1,2,Junting Yuan1,3,Tuoyu Geng1,2,Shihao Chen1,2,Xuming Hu1,2,Isabelle H. Cui6,Hengmi Cui1,2,4,5* From the 1Institute of Epigenetics and Epigenomics, 2College of Animal Science and Technology, 3College of Bioscience and Biotechology, 4Institute of Comparative Medicine,5Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu 225009, China;6Department of Pathology and Laboratory Medicine, New York Presbyterian-Weill Cornell Medicine, 525 East 68th Street, New York, New York 10065 USA Running title: gene silencing using PolyA signal via CRISPR/Cas9 system To whom correspondence should be addressed: Hengmi Cui, 86-514-87990309, and [email protected]. Keywords: CRISPR/Cas9, double marker selection, gene silencing, transcriptional terminator Downloaded from ABSTRACT The type II bacterial CRISPR/Cas9 sys- http://www.jbc.org/ tem is a simple, convenient and powerful tool Previous studies indicate that non-protein- for targeted gene editing. Here, we describe a coding genes, especially those that encode long CRISPR/Cas9-based approach for inserting a noncoding RNAs (lncRNAs), play a key role in PolyA transcriptional terminator into both alle- gene regulation and are involved in many biolog- les of a targeted gene to silence protein-coding ical processes, e.g., cell growth, epigenetic reg- by guest on May 8, 2020 and non-protein-coding genes, which often ulation, cancer development and human disease play key roles in gene regulation but are diffi- (1–4).Compared to protein-coding genes, silencing cult to silence via insertion or deletion of short long non protein-coding genes by insertion or dele- DNA fragments, The integration of a 225bp of tion of small DNA fragments is difficult as the bovine growth hormone (BGH) PolyA signal lncRNA function is primarily subject to conforma- into either the first intron or the first exon or tional changes. Recently, a CRISPR/Cas9-based behind promoter of target genes caused effi- system was developed as a novel tool for gene editing cient termination of expression of PPP1R12C, (5,6). As a simple, convenient and efficient system, NSUN2 (protein-coding genes) and MALAT1 it has been used for gene editing in a variety of (non-protein-coding gene). Both NeoR and organisms (7). The genes edited with this system in- PuroR were used as markers in the selection clude protein coding and non-protein coding genes. of clonal cell lines with biallelic integration Two strategies have been employed for permanently of a PolyA signal. Genotyping analysis indi- silencing non-protein-coding genes using large ge- cated that the cell lines displayed the desired nomic deletions with the CRISPR/Cas9 system. One biallelic silencing after a brief selection pe- strategy is to completely delete the given gene using riod. These combined results indicate that multiple guide RNAs (gRNAs) targeting the 5’ and this CRISPR/Cas9-based approach offers an 3’ flanking sequences (8–10). The alternative strat- easy, convenient and efficient novel technique egy is to delete core promoter sequences of the given for gene silencing in cell lines, especially for gene (11). The limitation of these strategies is that those, in which gene integration is difficult be- the deletion of a large genomic fragment may alter cause of a low efficiency of homology-directed the function of the interest gene due to removal of repair. the potential regulatory elements or other functional 1 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. gene silencing using PolyA signal via CRISPR/Cas9 system genes around the targeted genomic region. ing. In addition, multiple tandem PolyA signals were This study was designed to silence three shown to enhance transcriptional termination, and a genes, including lncRNA gene, MALAT1 by bial- beta-globin terminator has also been shown to ter- lelic integration of a PolyA signal using the minate the transcription driven by eukaryotic RNA CRISPR/Cas9 system and thus develop an easy, con- polymerase II (13, 14). Therefore, we constructed venient and efficient approach to silencing gene with a set of plasmids with the following cassettes: the advantages of both the CRISPR/Cas9 and PolyA CMV promoter-RFP-IRES -EGFP (for the control signal approaches. Firstly, a PolyA signal was op- plasmid), CMV promoter-RFP-BGH PolyA signal- tionally inserted into three designated sites (imme- IRES-EGFP, CMV promoter-RFP-4× BGH PolyA diately behind the promoter, at the first exon or the signals-IRES-EGFP and CMV promoter-RFP- beta- first intron) of the targeted gene via CRISPR /Cas9- globin terminator-IRES-EGFP (Figure 1A). Red flu- induced homology-directed repair (HDR). Secondly, orescent protein (RFP) and enhanced green fluores- double marker selection was employed for screen- cent protein (EGFP) were used as selection mark- ing clonal cell lines with successful biallelic inte- ers. All plasmids were independently transfected gration of the PolyA signal. Lastly, the efficiency into HEK293 cells. After 48 hours of transfection, of gene silencing was evaluated by qRT-PCR, and the transcription of sequences downstream the BGH biallelic integration was verified by genotyping anal- PolyA signal, 4× BGH PolyA signals and the beta- Downloaded from ysis. Our data showed that the transcription of the globin terminator was drastically reduced compared given gene was efficiently terminated, demonstrat- to controls, as indicated by the qRT-PCR analysis ing that CRISPR/Cas9-mediated biallelic integra- (Figure 1B). Moreover, the effect of the beta-globin tion of a PolyA signal with double marker selection terminator on gene silencing was stronger than that http://www.jbc.org/ is an easy, convenient and efficient novel approach of the BGH PolyA signal and multiple tandem BGH for gene silencing in cell line. PolyA signals lead to an enhanced transcriptional termination. RESULTS To determine if the terminators driven by Downstream transcription was sup- an endogenous promoter could silence the targeted by guest on May 8, 2020 pressed by transcriptional terminators immedi- gene, we constructed a set of plasmids similar to the ately behind an endogenous promoter. A vi- ones described above. The differences of the plas- ral promoter (CMV promoter) was used for driv- mids are: 1. The CMV promoter was replaced by the ing the transcription of the selection marker gene endogenous EF-1A promoter; 2. The RFP gene was (GFP) and a PolyA signal in a previous study, in removed; and 3. An EF-1A promoter-reversed BGH which a ZNF-based approach to gene silencing was PolyA signal-IRES-EGFP cassette was constructed established(12). Here, we tested whether an en- to test whether the integration of BGH PolyA sig- dogenous promoter could do the same as the viral nal had any effect on antisense transcription (Fig- promoter with the following specific aims: To test ure 2A). The qRT-PCR analysis with primers spe- whether an integration construct could be driven by cific for IRES showed that, compared to the con- the promoter of the targeted gene so that the inte- trol, the transcription downstream the BGH PolyA gration construct can be shortened to facilitate its signal, 4× BGH PolyA signals and the beta-globin transfection into cells without an exogenous pro- terminator was drastically inhibited after transfec- moter; Because viral promoters are easily subject tion with the individual plasmid into HKE293 cells. to epigenetic modification in eukaryotic cells, an An enhanced inhibition of downstream transcription endogenous promoter may allow a more persistent was observed in the cells transfected with the plas- transcription of the integration construct. mid containing 4× BGH PolyA signals. In contrast, We also verified whether a PolyA signal and transfection with the plasmid containing a reversed beta-globin terminator could terminate downstream BGH PolyA signal did not cause significant inhi- transcription in our system using a CMV promoter. bition of its downstream transcription (Figure 2A). The BGH PolyA signal was previously used as an Red fluorescent protein (RFP) and enhanced green RNA destabilizing element (RDE) for gene silenc- fluorescent protein (EGFP) were used as selection 2 gene silencing using PolyA signal via CRISPR/Cas9 system markers. All plasmids were independently trans- (Table 1). Moreover, as the PuroR selection marker fected into HEK293 cells. After 48 hours of trans- lacked a promoter in the donor plasmid, 4 out of 62 fection, the transcription of sequences downstream (6.5%) clonal lines with the PuroR marker randomly the BGH PolyA signal, 4× BGH PolyA signals and integrated into the genome. Using the cell lines with the beta-globin terminator was drastically reduced the PolyA signal integration, we determined whether compared to controls, as indicated by the qRT-PCR the PPP1R1 gene was prematurely terminated as a analysis (Figure 2B), suggesting that the integration result of the integration. The results from the qRT- of the BGH PolyA signal had a negligible effect on PCR analysis with two pairs of primers specific for the antisense transcription. This feature may be very PPP1R12C gene revealed that, compared to the con- useful for avoiding interference with the transcrip- trol, the expression of PPP1R12C was drastically re- tion of an overlapping antisense gene on the comple- duced by the integration of the PuroR-BGH-PolyA mentary DNA strand, as this may potentially cause (Figure 3D), suggesting that biallelic integration of a misinterpretation of the function of the targeted the PolyA signal via CRISPR/Cas9-induced HDR gene (15).
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