gene silencing using PolyA signal via CRISPR/Cas9 system

Biallelic Insertion of a Transcriptional Terminator via CRISPR/Cas9 Efficiently Silences Expression ofProtein-coding and Non-coding RNA Genes

Yangyang Liu1,2, Xiao Han1,2,Junting Yuan1,3,Tuoyu Geng1,2,Shihao Chen1,2,Xuming Hu1,2,IsabelleH. 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-innovationCenter for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou

University, Yangzhou, Jiangsu 225009, China;6Department of Pathology and Laboratory Medicine, NewYork 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 hmcui@yzu.edu.cn.

Keywords: CRISPR/Cas9, double marker selection, gene silencing, transcriptional terminator

ABSTRACTThe type II bacterial CRISPR/Cas9 sys-

tem is a simple, convenient and powerful toolfor targeted gene editing. Here, we describe aCRISPR/Cas9-based approach for inserting aPolyA transcriptional terminator into both alle-les of a targeted gene to silence protein-codingand non-protein-coding genes, which oftenplay key roles in gene regulation but are diffi-cult to silence via insertion or deletion of shortDNA fragments, The integration of a 225bp ofbovine growth hormone (BGH) PolyA signalinto either the first intron or the first exon orbehind promoter of target genes caused effi-cient termination of expression of PPP1R12C,NSUN2 (protein-coding genes) and MALAT1(non-protein-coding gene). Both NeoR andPuroR were used as markers in the selectionof clonal cell lines with biallelic integrationof a PolyA signal. Genotyping analysis indi-cated that the cell lines displayed the desiredbiallelic silencing after a brief selection pe-riod. These combined results indicate thatthis CRISPR/Cas9-based approach offers aneasy, convenient and efficient novel techniquefor gene silencing in cell lines, especially forthose, in which gene integration is difficult be-cause of a low efficiency of homology-directedrepair.

Previous studies indicate that non-protein-coding genes, especially those that encode longnoncoding RNAs (lncRNAs), play a key role ingene regulation and are involved in many biolog-ical processes, e.g., cell growth, epigenetic reg-ulation, cancer development and human disease(1–4).Compared to protein-coding genes, silencinglong non protein-coding genes by insertion or dele-tion of small DNA fragments is difficult as thelncRNA function is primarily subject to conforma-tional changes. Recently, a CRISPR/Cas9-basedsystemwas 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 oforganisms (7). The genes edited with this system in-clude protein coding and non-protein coding genes.Two strategies have been employed for permanentlysilencing non-protein-coding genes using large ge-nomic deletionswith theCRISPR/Cas9 system. Onestrategy is to completely delete the given gene usingmultiple guide RNAs (gRNAs) targeting the 5’ and3’ flanking sequences (8–10). The alternative strat-egy is to delete core promoter sequences of the givengene (11). The limitation of these strategies is thatthe deletion of a large genomic fragment may alterthe function of the interest gene due to removal ofthe potential regulatory elements or other functional

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http://www.jbc.org/cgi/doi/10.1074/jbc.M116.769034The latest version is at JBC Papers in Press. Published on February 14, 2017 as Manuscript M116.769034

Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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genes around the targeted genomic region.This study was designed to silence three

genes, including lncRNA gene, MALAT1 by bial-lelic integration of a PolyA signal using theCRISPR/Cas9 system and thus develop an easy, con-venient and efficient approach to silencing gene withthe advantages of both the CRISPR/Cas9 and PolyAsignal approaches. Firstly, a PolyA signal was op-tionally inserted into three designated sites (imme-diately behind the promoter, at the first exon or thefirst intron) of the targeted gene via CRISPR /Cas9-induced homology-directed repair (HDR). Secondly,double marker selection was employed for screen-ing clonal cell lines with successful biallelic inte-gration of the PolyA signal. Lastly, the efficiencyof gene silencing was evaluated by qRT-PCR, andbiallelic integrationwas verified by genotyping anal-ysis. Our data showed that the transcription of thegiven gene was efficiently terminated, demonstrat-ing that CRISPR/Cas9-mediated biallelic integra-tion of a PolyA signal with double marker selectionis an easy, convenient and efficient novel approachfor gene silencing in cell line.

RESULTSDownstream transcription was sup-

pressed by transcriptional terminators immedi-ately behind an endogenous promoter. A vi-ral promoter (CMV promoter) was used for driv-ing the transcription of the selection marker gene(GFP) and a PolyA signal in a previous study, inwhich a ZNF-based approach to gene silencing wasestablished(12). Here, we tested whether an en-dogenous promoter could do the same as the viralpromoter with the following specific aims: To testwhether an integration construct could be driven bythe promoter of the targeted gene so that the inte-gration construct can be shortened to facilitate itstransfection into cells without an exogenous pro-moter; Because viral promoters are easily subjectto epigenetic modification in eukaryotic cells, anendogenous promoter may allow a more persistenttranscription of the integration construct.

We also verified whether a PolyA signal andbeta-globin terminator could terminate downstreamtranscription in our system using a CMV promoter.The BGH PolyA signal was previously used as anRNA destabilizing element (RDE) for gene silenc-

ing. In addition, multiple tandemPolyA signalswereshown to enhance transcriptional termination, and abeta-globin terminator has also been shown to ter-minate the transcription driven by eukaryotic RNApolymerase II (13, 14). Therefore, we constructeda set of plasmids with the following cassettes:CMV promoter-RFP-IRES -EGFP (for the controlplasmid), CMV promoter-RFP-BGH PolyA signal-IRES-EGFP, CMV promoter-RFP-4× BGH PolyAsignals-IRES-EGFP and CMV promoter-RFP- beta-globin terminator-IRES-EGFP (Figure 1A). Red flu-orescent protein (RFP) and enhanced green fluores-cent protein (EGFP) were used as selection mark-ers. All plasmids were independently transfectedinto HEK293 cells. After 48 hours of transfection,the transcription of sequences downstream the BGHPolyA signal, 4× BGH PolyA signals and the beta-globin terminator was drastically reduced comparedto controls, as indicated by the qRT-PCR analysis(Figure 1B). Moreover, the effect of the beta-globinterminator on gene silencing was stronger than thatof the BGH PolyA signal and multiple tandem BGHPolyA signals lead to an enhanced transcriptionaltermination.

To determine if the terminators driven byan endogenous promoter could silence the targetedgene, we constructed a set of plasmids similar to theones described above. The differences of the plas-mids are: 1. TheCMVpromoter was replaced by theendogenous EF-1A promoter; 2. The RFP gene wasremoved; and 3. An EF-1A promoter-reversed BGHPolyA signal-IRES-EGFP cassette was constructedto test whether the integration of BGH PolyA sig-nal had any effect on antisense transcription (Fig-ure 2A). The qRT-PCR analysis with primers spe-cific for IRES showed that, compared to the con-trol, the transcription downstream the BGH PolyAsignal, 4× BGH PolyA signals and the beta-globinterminator was drastically inhibited after transfec-tion with the individual plasmid into HKE293 cells.An enhanced inhibition of downstream transcriptionwas observed in the cells transfected with the plas-mid containing 4× BGH PolyA signals. In contrast,transfection with the plasmid containing a reversedBGH PolyA signal did not cause significant inhi-bition of its downstream transcription (Figure 2A).Red fluorescent protein (RFP) and enhanced greenfluorescent protein (EGFP) were used as selection

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markers. All plasmids were independently trans-fected into HEK293 cells. After 48 hours of trans-fection, the transcription of sequences downstreamthe BGH PolyA signal, 4× BGH PolyA signals andthe beta-globin terminator was drastically reducedcompared to controls, as indicated by the qRT-PCRanalysis (Figure 2B), suggesting that the integrationof the BGH PolyA signal had a negligible effect onthe antisense transcription. This feature may be veryuseful for avoiding interference with the transcrip-tion of an overlapping antisense gene on the comple-mentary DNA strand, as this may potentially causea misinterpretation of the function of the targetedgene (15).

Taken together, these findings indicated thatthe BGH PolyA signal driven by an endogenous pro-moter was able to efficiently terminate its down-stream transcription. Because BGH PolyA is a shortterminator, its integration into a target gene maybe more efficiently mediated by a CRISPR/Cas9-induced HDR approach than by other long termina-tors (i.e., 4× BGH PolyA signals and the beta-globinterminator). We thus speculated that a biallelicBGH PolyA integration mediated by CRISPR/Cas9-induced HDR could efficiently shut down transcrip-tion of a target gene.

The transcription of the PPP1R12C genewas terminated by an integrated BGH PolyA sig-nal. To silence the PPP1R12C gene, a protein-coding gene, by biallelic integration of a PolyA sig-nal via CRISPR/Cas9-induced HDR, a donor plas-mid was constructed for integrating a PolyA sig-nal into the adeno-associated virus integration site 1(AAVS1) in the first intron of the PPP1R12C genebased on gene trapping strategy (Figure 3A). AAVS1is a “safe harbor” gene located within the first in-tron of the human PPP1R12C gene citemali2013rna.This plasmid contains a splice adopter (SA), T2A, apuromycin resistance (PuroR) selection marker anda BGH PolyA signal, flanked by homologous se-quences of AAVS1 gene (Figure 3B). The donorplasmid was co-transfected into HEK293 cells withthe plasmids expressing the Cas9 protein and theAAVS1 guide RNA (gRNA). A PCR-based geno-typing analysis using genomic DNA revealed that93.5% of clonal cell lines were transgenic after 2weeks of puromycin selection, with only 3 out of62 (4.9%) clonal lines having biallelic transgenes

(Table 1). Moreover, as the PuroR selection markerlacked a promoter in the donor plasmid, 4 out of 62(6.5%) clonal lines with the PuroRmarker randomlyintegrated into the genome. Using the cell lines withthe PolyA signal integration, we determinedwhetherthe PPP1R1 gene was prematurely terminated as aresult of the integration. The results from the qRT-PCR analysis with two pairs of primers specific forPPP1R12C gene revealed that, compared to the con-trol, the expression of PPP1R12C was drastically re-duced by the integration of the PuroR-BGH-PolyA(Figure 3D), suggesting that biallelic integration ofthe PolyA signal via CRISPR/Cas9-induced HDRefficiently silenced the transcription of its target geneeven if the integration took place in the intron of thegene.

Efficient selection for biallelic silencingby double selection markers. A single markerselection for PolyA signal integration was not ef-ficient and only 4.9% of the clonal cell lines showedbiallelic integration of the PolyA signal into thePPP1R12C gene (Table 1). This low efficiencymay become worse if genes do not respond wellto Cas9/gRNA or HDR (11). We therefore testedwhether double selection markers (e.g., neomycinresistance (NeoR) and PuroR for drug selection)could improve the efficiency of the biallelic silenc-ing. The plasmid with NeoR-BGH PolyA cassettewas constructed in the same way as the plasmidwith PuroR-BGH PolyA cassette (Figure 4A). Thetwo plasmids were co-transfected into HEK293 cellswith the Cas9/AAVS1-gRNAplasmids. Genotypinganalysis showed that, after two weeks of double drugselection, 94.2% of clonal cell lines were bialleli-cally transgenic, and only 4 out of 69 (5.8%) clonalcell lines were monoallelically transgenic ((Figure4C), Table 1). The biallelic integration of the PolyAsignal was mediated by Cas9/gRNA-induced HDR(Figure 4B). Moreover, qRT-PCR analysis on theclonal lines revealed that the transcription of thePPP1R12C gene was dramatically shut down (Fig-ure 4D). Together, these findings indicated that bial-lelic silencing was largely improved by the doublemarker selection.

Silencing the lncRNA gene by biallelic in-tegration of a PolyA signal. MALAT1 is a longnoncoding RNA (lncRNA) gene that is highly ex-pressed in a variety of cell lines (e.g., A549, Hela,

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and HepG2). Because silencing lncRNA is consid-ered a challenge, we tested whether the approachused to silence the PPP1R12C gene as describedabove could also be applied to lncRNA genes. Forthis purpose, two donor plasmids were constructedcontaining a 5’-homologous arm (HA), PuroR (orNeoR), the BGH PolyA signal, and a 3’-HA (Figure5A). A plasmid containing gRNA targeting to the se-quences immediately behind theMALAT1 promoterwas also constructed. These donor plasmids did notinclude SA-T2A sequences as MALAT1 is a non-protein- coding gene. These plasmids, together withthe Cas9-containing plasmid, were co-transfectedinto HepG2 cells. After two weeks of double drugselection, 37 out of 47 (78.7%) clonal cell lines werebiallelically transgenic with the insertion of NeoR(or PuroR) and theBGHPolyA signal sequences (Ta-ble 1), as indicated by the genotyping analysis usingspecific primers (MALAT1-HDR-F/R) for the trans-genic fragments (Figure 5B). The qRT-PCR analysisshowed that the mRNA abundance of MALAT1 inthe cellswith biallelic integration of the PolyA signalwas reduced to 0.1% compared to the control cells(Figure 5C). These findings suggested that biallelicintegration of the PolyA signal via CRISPR/Cas9-induced HDR efficiently silenced the transcriptionof a long non-protein-coding gene, MALAT1.

Silencing NSUN2 by biallelic integrationof a PolyA signal. We also tested whether biallelicintegration of a PolyA signal into the open readingframe of the NSUN2 gene, a RNA methyltransfersegene, could disrupt the transcription of this protein-coding gene. Similarly, two donor plasmids wereconstructed containing a 5’-HA, T2A, PuroR (orNeoR), the BGH PolyA signal, and a 3’-HA (Figure6A). The plasmid containing gRNA targeting to thefirst exon of the NSUN2 gene was also constructed.These plasmids, together with the Cas9-containingplasmid, were co-transfected into HEK293 cells.After two weeks of double drug selection, 17 out of26 (65.4%) clonal cell lines were biallelically trans-genic (Table 1), as indicated by genotyping anal-ysis using specific primers (NSUN2-HDR-F/R) forthe transgenic fragments (Figure 6B). The qRT-PCRanalysis showed the expression of NSUN2 in thecells with biallelic integration of PolyA signal wasreduced to 1-3% compared to the control cells (Fig-ure 6C). These results suggested that biallelic inte-

gration of a PolyA signal into the first exon of theNSUN2 gene efficiently terminated the transcriptionof this protein-coding gene.

DISCUSSIONGene deletion or knockout is a useful tool

for gene functional studies. However, it is often nec-essary to remove the whole sequence of the targetedgene or a large DNA fragment surrounding the pro-moter or the whole gene (8, 11). This can causea misinterpretation of the function of the targetedgene because it is possible that a gene on the com-plimentary DNA strand overlaps with the targetedgene and/or its regulatory sequence or that a partialcoding sequence is also removed due to this over-lap (e.g., the genomic region that can be transcribedinto sense and antisense transcripts). RNA interfer-ence is another useful tool commonly used for genesilencing, but it also has several shortcomings, in-cluding a high off-targeting rate and insufficient genesilencing (16).

In this study, we developed a novel approachto gene silencing using the biallelic integration ofa PolyA signal mediated by CRISPR/Cas9-inducedHDR. The CRISPR/Cas9 system is a tool for geneediting that has been recently developed for geneknockout and knock-in (5, 17) . Compared to othergene editing tools, it is simple, easy and convenientand has the potential to target multiple genes si-multaneously. More importantly, the CRISPR/Cas9system can significantly increase the rate of HDR-mediated biallelic integration (5, 18). In this study,we leveraged the power of the CRISPR/Cas9 systemand used a BGH PolyA signal together with two se-lection markers to successfully integrate constructsinto PPP1R12C, NSUN2, andMALAT1 at three dif-ferent sites (i.e., the first intron of PPP1R12C, thefirst exon of NSUN2, and directly behind the pro-moter of MALAT1). Because this approach doesnot involve either the removal of large DNA frag-ments or RNA interference, it overcomes some ofthe shortcomings of the conventional gene editingtools such as the low efficiency of HDR-mediatedbiallelic integration and the extensive screening ofclonal cells (4, 18). In addition, the ability to choosefrom multiple integration sites allows the side ef-fects of the integrated PolyA signal in the targetedor overlapping genes to be prevented. Moreover,

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as indicated by qRT-PCR analysis, gene silencingby biallelic integration of a PolyA signal did notcompletely block the transcription of the targetedgene. This feature makes the novel approach espe-cially useful for functional studies of genes whosedeletions are lethal.

The successful integration of the BGHPolyA signal significantly shut down the transcrip-tion of three genes, including two protein-codinggenes (PPP1R12C and NSUN2) and a long non-protein-coding gene, MALAT1. Although it is notsurprising that the protein-coding genes were si-lenced through the novel approach, the almost com-plete shutdown of the long non protein-coding geneimplies that this approach can be applied for silenc-ing almost any gene of interest.

In this study, the efficiency of gene silenc-ing by biallelic integration of a PolyA signal wasgreatly improved by double marker selection. Al-though the marker genes were driven by the endoge-nous promoter of the targeted gene, expression ofthe marker genes appeared to be sufficient for effec-tive selection. The results from the single markerselection study showed that only 4.8% of clonal celllines were biallelically transgenic. In contrast, thedouble marker selection lead to 65-93% of biallelicintegration by two-week selection. Based on theseresults, we can speculate that double marker selec-tion may be particularly useful for the cell lines withlow biallelic integration efficiency. If we could pro-long antibiotics selection time (e.g. from two weeksto three weeks, we would have much better biallelictransgenic rates. Nevertheless, if the endogenouspromoter of the targeted gene was very weak, thisapproach would not be appropriate because the ex-pression of marker genes may not be sufficient fordrug selection. For example, the expression of theHOTAIR gene in HEK293 cells was very low (Fig-ure 7)and under this promoter, the integrated markergene had a very low expression, leading to the failureof drug selection (data not shown). In this case, anadditional promoter (e.g., EF-1A) may be insertedin front of the marker gene to drive its expression(Figure 8) (12). In conclusion, we tried three dif-ferent insertion targets for PolyA integration, i.e., anintegration site within the first intron, within the firstexon, or immediately behind the promoter of a tar-get gene. These strategies provide multiple choices

for PolyA integration sites to achieve the best si-lencing efficiency based on the specificity of a targetgene. Using this approach, we can establish a cellline with a coding or non-coding gene silencing veryefficiently and shortly.

EXPERIMENTAL PROCEDURESCell Culture and Transfection. The hu-

man embryonic kidney cell line 293 (HEK293) waspurchased from ATCC (CRL-1573, USA). Cellswere maintained in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% fetalbovine serum (HyClone, USA), 2 mM GlutaMAX(Life Technologies, USA), 100 U/ml penicillin and100 mg/ml streptomycin at 37°C with 5% CO2. Fortransfection, cells were seeded into 60 mm dishes(Corning, USA) at a density of 2×106 cells/dishand cultured in an antibiotic-free medium. Whencells were at 80%–90% confluency, they were trans-fectedwith plasmids usingLipofectamine 2000 (LifeTechnologies, USA) according to themanufacturer’sinstructions. For PolyA testing, cells were trans-fected with a plasmid mixture (a total 4 µg of BGH-puroR and BGH-neoR donor plasmids and 4 µg ofCas9/sgRNA plasmid). After 2 days of transfec-tion, cells were treated with 1 µg/mL of Puromycin(Sigma, USA) for 3 days, and then followed by treat-ment with 400 µg/ml of Neomycin (Sigma, USA) for14 days.

Construction of PolyA-Contained Plas-mids. The BGH PolyA signal was amplified froma pcDNA3.1 (+) vector by PCR. The 4 × PolyAwas constructed with 4 PolyAs using a Golden gateclone. The beta-globin PolyA was also amplifiedfrom HEK293 cell genomic DNA following previ-ous published protocols (14). Four different PolyAsignals were inserted into the EcoRI site of pIRES2-EGFP (Clontech, USA), followed by the replacementof the CMV promoter with the EF-1A promoter.The four vectors containing the viral CMV promot-ers were digested with BglII and ligated with theRFP gene derived from pGBT-RP2-1. A list of allcloning primers are listed in the Supplemental TableS1.

Construction of vectors. The sgRNA se-quences were synthesized and annealed to the Cas9expression plasmid (Addgene ID 42230, USA) ac-cording to a previously published protocol (19). The

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Neo-BGH poly (A) donor was constructed as previ-ously described (20, 21). In brief, the NeoR andPuroR genes were subcloned from the pcDNA3.1(+) vector (Invitrogen) and the pSMPUW-IRES-Puro vector (Cell Biolabs, USA), respectively. TheNeoR and PuroR genes were then linked with BGHPolyA signals through overlapping PCR. The leftand right homology sequences were cloned fromHEK293 cell genomic DNA. The pGEM®-T EasyVector (Promega, city, country) was amplified witha pair of oligo primers containing the BsmbI re-striction site and used as a vector backbone. Togenerate NeoR/PuroR-BGH poly (A) donor vec-tors, NeoR/PuroR-BGH poly (A) and homologysequences generated by amplification with BbsIsite-containing oligo primers were cloned into theBbsI-digested vector using the Golden Gate cloningmethod.

Reverse transcription and qPCR. Cellswere disrupted in TRIzol (Life Tech, city, country)and RNA was extracted according to the manufac-turer’s instructions. Complementary DNAs weresynthesized using the PrimeScriptTM RT reagentKit plus gDNA Eraser (Takara, city, country) andrandom primers. Quantitative PCR was performedusing SYBR Premix Ex Taq (Takara, DDR420A,city, country). Regular PCR was performed usingTaq polymerase (Fermentas, city, country) followingmanufacturer’s recommendations.

Isolation of genomic DNA and PCR-based genotyping analysis. Cells in 96-well plateswere lysed with 0.1 ml lysis buffer containing 10mM Tris pH 8, 2 mM EDTA, 0.2% Triton X-100,and 200 µg/mL Proteinase K. After 2 hours of incu-bating at 50-56°C, the cells were heated at 95°C for

5 min to inactivate the proteinase K. Five ml samplewere used for a subsequent integration-oriented PCRwith 2 ×TaqMasterMixture (Vazyme, city, country)according to the manufacturer’s recommendations.

Luciferase Reporter Assays for PromoterActivity. The reporter vector of pGL3 (Promega,city, country) was used for transfection into HEK293cells. We first generated several promoter sequencesby PCRwith the primers listed in Table S1. The frag-ments included the MALAT1 large promoter (nu-cleotides +45 to -1047bp, of the TSS), the HDR-MALAT1 promoter (nucleotides +45 to -729bp),and the HOTAIR promoter (nucleotides +204 to-904bp of the TSS in NR047518.1). These pro-moter fragments were respectively cloned into thepGL3-promoter vector using two cloning sites (XhoIandHindIII). The constructs were namedMALAT1-large, MALAT1-small, HDR-MALAT1 and pGL3-Hotair, respectively. HEK293 cells were seeded ata density of 4 × 105 cells per well in 12-well plates24 hours before transfection. Four hundred ng lu-ciferase plasmid containing different promoterswereco-transfected with 2 ng of pRL-TK plasmid. ThePGL3-control and PGL3-Basic constructs were usedas positive and negative controls, respectively. TheLipofectamine® 2000 transfection reagent (Invitro-gen, USA) was used. Forty-eight hours after trans-fection, the luciferase activitywasmeasured and nor-malized.

FACS analysis. HEK293 cells were trans-fected with the plasmid containing the EGFP andRFP genes. Cells were then analyzed on a FACSAria II cell sorter (BD Biosciences, USA) after 24hours of incubation. All primer sequence informa-tion in the experiment can be obtained by inquiry.

Acknowledgments: This research was supported by the National Key Research and Development Programin China (2016YFC1303604), the National Natural Science Foundation of China (81171965, 81372237and 91540117), and the Priority Academic Program Development of Jiangsu Higher Education Institutions(Animal Science and Veterinary Medicine).

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of thisarticle.

Author contributions: HC and YL conceived of the study and participated in its design and coordination.YL, XH, JY XH and CS carried out the experiments. HC, YL, IHC and TG drafted the manuscript. Allauthors read and approved the final manuscript.

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21. González, F., Zhu, Z., Shi, Z.-D., Lelli, K., Verma, N., Li, Q. V., and Huangfu, D. (2014) An icrisprplatform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells,Cell stem cell 15(2), 215–226.

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gene silencing using PolyA signal via CRISPR/Cas9 system

FOOTNOTES5’HA: 5’ left arm homologous, CMV promoter: cytomegalovirus enhancer and myeloproliferative sarcomavirus promoter, PuroR: puromycin resistancegene, NeoR: neomycin resistancegene, BGH polyA: bovinegrowth hormone polyadenylation, 3’HA : 3’ right arm homologous.

TABLES

Silenced gene Integration site Selection marker Total No HDR Homozygous Heterozygous All HDRPPP1R12C Intron 1 PuroR 62 4(6.3%) 3(4.9%) 55(88.7%) 58(93.5%)PPP1R12C Intron 1 PuroR+NeoR 69 0(0) 65(94.2%) 4(5.8%) 125(100%)MALAT1 near promoter PuroR+NeoR 47 4(8.5%) 37(78.7%) 8(17%) 48(91.4%)NSUN2 Exon 1 PuroR+NeoR 26 3(11.5%) 17(65.4%) 6(23.1%) 23(88.8%)

Table 1: The efficiency of gene silencing using polyA signal integration

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FIGURES

Figure 1: Analysis of silencing potency for different silencing signals driven by viral promoter.(A) Thestructures of different plasmids constructed for this analysis. The control plasmid is highlighted with a viralpromoter, CMV promoter (blue arrow), RFP (red box), IRES (orange box) and EGFP (green box).The otherplasmids are constructed from control plasmid. The difference of the plasmids from control plasmid is thedifferent silencing signals inserted between RFP and IRES, including BGH PolyA(+) (black arrow),4xBGHPolyA (4 black arrows), and beta-globin terminator (long black arrow).

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gene silencing using PolyA signal via CRISPR/Cas9 system

Figure 2: Analysis of silencing potency for different terminator signals driven by an endogenousEF-1A promoter.(A) The structures of plasmids with different terminator signals. The control plasmidconsists of an endogenous promoter, EF-1A promoter (blue arrow), IRES (orange box) and EGFP (greenbox) without terminator signal. The other plasmids are constructed based on the control plasmid with thedifferent terminator signals inserted between EF-1A promoter and IRES, including BGH polyA(+) (a blackarrow), BGH polyA(-) (a reverse black arrow), 4xBGH PolyA(4 black arrows), and beta-globin terminator(a long black arrow).(B) qRT-PCR analysis with primers (IRES-F/R) of HEK293 cells transfected withdifferent plasmids containing different terminator signals. Compared to control plasmid, the other plasmidsefficiently silenced the transcription except for BGH PolyA(-) plasmid. The transcription is presented as foldchanges over the control plasmid (N=3). The data are presented as the means + standard deviation (SD).BGH PolyA(+), bovine growth hormone polyadenylation sequences; BGH PolyA(-), reversed BGH PolyAsequences; IRES, internal ribosomal entry site sequences, EGFP: enhanced green fluorescent protein gene.

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gene silencing using PolyA signal via CRISPR/Cas9 system

Figure 3: Crispr/Cas9-based HDR-mediated biallelic integration of polyA into the first intron ofPPP1R12C or the site of AAVS1 causes downstream transcriptional termination.(A) A schematicoverview of PPP1R12C transcript where the positions of AAVS1 gene and gRNA targeting to it are shown.(B)Schematic diagram illustrating the strategy for PPP1R12C gene silencing through homology directed repair(HDR) approach. The donor plasmid contains left and right arms (5’-and 3’-HA) homologous to AAVS1,SA-T2A, PuroR, and BGH polyA. (C) PCR-based genotyping analysis for identifying clonal cell lineswith wild type of PPP1R12C, or with monoallelic/biallelic integration of polyA signal into the first intron ofPPP1R12C or the site of AAVS1 gene. Primers used for genotyping analysis are named as AAVS1-HDR-F/R,which specifically bind to AAVS1 at left and right homologous arms (5’-and 3’-HA). The gel image at thebottom showingwild type is homozygous for PPP1R12C gene as indicated by 1976bp single amplicon, clonalcell line 17 and 23 are both homozygous for biallelically integrated polyA signals as indicated by 3310bpsingle amplicon, and clonal cell line 20 is heterozygous as indicated by 1976bp and 3310bp amplicons.(D) qRT-PCR analysis with two sets of primers (1/2Exon-F/R, a set for exon 1 and 2 of PPP1R12C, and3/4Exon-F/R, a set for exon 3 and 4 of PPP1R12C) indicates that, compared to HEK293 cells transfectedwith control plasmid, the abundance of the targeted transcripts is drastically reduced. GAPDH is used asinternal control. N=3. The data are presented as the means + standard deviation (SD).

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gene silencing using PolyA signal via CRISPR/Cas9 system

Figure 4: Crispr/Cas9-based gene silencing by biallelic integration of polyA signals is efficiently im-proved with the use of double selection markers.(A) A schematic overview of donor plasmids constructedfor double drug selection. (B) PCR-based genotyping analysis for identifying clonal cell lines with wild typeof PPP1R12C, or with biallelic integration of polyA signal into the first intron of PPP1R12C or the site ofAAVS1 gene. Primers used for genotyping analysis are named as AAVS1-HDR-F/R, which specifically bindto AAVS1 at left and right homologous arms (5’-and 3’-HA). (C) The gel image wild type is homozygous forPPP1R12C gene as indicated by 1976bp single amplicon, clonal cell lines 6, 8, 15 and 16 are all homozygousfor biallelically integrated polyA signals as indicated by 3310bp (PuroR) and 3654bp amplicons. (D) qRT-PCR analysis with primers (3/4Exon-F/R, a set for exon 3 and 4 of PPP1R12C) indicates that, compared toHepG2 cells transfected with control plasmid, the abundance of the targeted transcript is drastically reduced.GAPDH is used as internal control. N=3. The data are presented as the means + standard deviation (SD).

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Figure 5: Crispr/Cas9-based HDR-mediated biallelic integration of polyA into the site just behindthe promoter of MALAT1 causes downstream transcriptional termination.(A) Schematic diagram il-lustrating the strategy for MALAT1 gene silencing through homology directed repair (HDR) approach. Thedonor plasmids contain left and right arms (5’-and 3’-HA) homologous to MALAT1, PuroR/NeoR, andBGH polyA. The region in yellow denotes promoter, which partially overlaps with 5’-HA. 5’HA: 5’ left armhomologous to MALAT1, PuroR: puromycin resistancegene, NeoR: neomycin resistancegene, BGH polyA:bovine growth hormone polyadenylation, 3’HA : 3’ right arm homologous to MALAT1. (B) PCR-basedgenotyping analysis for identifying clonal cell lines with wild type of MALAT1, or with biallelic integrationof polyA signal into the site just behind the promoter ofMALAT1 gene. Primers used for genotyping analysisare named as MALAT1-HDR-F/R, which specifically bind to MALAT1 at left and right homologous arms(5’-and 3’-HA). The gel image showing wild type is homozygous for MALAT1 gene as indicated by 1831bpsingle amplicon, clonal cell lines 2, 3, 5 and 7 are all homozygous for biallelically integrated polyA signalsas indicated by 2589bp (PuroR) and 2734bp amplicons. (C) qRT-PCR analysis with primers (MALAT1-HDR-F/R) indicates that, compared to HepG2 cells transfected with control plasmid, the abundance of thetargeted transcript is drastically reduced. GAPDH is used as internal control. N=4. The data are presentedas the means + standard deviation (SD).

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gene silencing using PolyA signal via CRISPR/Cas9 system

Figure 6: Crispr/Cas9-based HDR-mediated biallelic integration of polyA into the first exon ofNSUN2 causes downstream transcriptional termination.(A) Schematic diagram illustrating the strat-egy for NSUN2 gene silencing through homology directed repair (HDR) approach. The donor plasmidscontain left and right arms (5’-and 3’-HA) homologous to NSUN2 exon1, T2A, PuroR/NeoR, and BGHpolyA. The sequence of gRNA (in red) against NSUN2 is shown. 5’HA: 5’ left arm homologous to NSUN2exon1, T2A: self-cleaving2A peptide, PuroR: puromycin resistancegene, NeoR: neomycin resistancegene,BGH polyA: bovine growth hormone polyadenylation, 3’HA: right arm homologous to NSUN2 exon1. (B)PCR-based genotyping analysis for identifying clonal cell lines with wild type of NSUN2, or with biallelicintegration of polyA signal into the first exon of NSUN2 gene. Primers used for genotyping analysis arenamed as NSUN2-HDR-F/R, which specifically bind to NSUN2 at left and right homologous arms (5’-and3’-HA). The gel image showing wild type is homozygous for NSUN2 gene as indicated by 1981bp singleamplicon, clonal cell lines 2, 3, and 5 are all homozygous for biallelically integrated polyA signals as indi-cated by 2739bp (PuroR) and 2884bp amplicons. (C) qRT-PCR analysis with primers (NSUN2-HDR-F/R)indicates that, compared to HEK293 cells transfected with control plasmid, the abundance of the targetedtranscript is drastically reduced. GAPDH is used as internal control. N=4. The data are presented as themeans + standard deviation (SD).

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gene silencing using PolyA signal via CRISPR/Cas9 system

Figure 7: Luciferase assays showing the activities of different promoters constructed in pGL3 plas-mid.The plasmid without any promoter, pGL3-Basic, is used as a negative control. The pGL3-controlplasmid, which contains a control promoter sequence, is used as a positive control. The other plasmids,containingMALAT1 promoter, HDR-MALAT1 promoter, PPP1R12C promoter, or Hotair promoter, are con-structed from pGL3-control. HDR sequence in pGL3-HDR-MALAT1 denotes the sequence homologous toMALAT1 is constructed into plasmid for homology-directed repair (HDR).

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gene silencing using PolyA signal via CRISPR/Cas9 system

Figure 8: Schematic illustration of the Crispr/Cas9-based strategy for Hotair gene silencing by biallelicintegration of polyA signal through homology directed repair (HDR) approach.The donor plasmidcontains left and right arms (5’-and 3’-HA) homologous to Hotair, CMV promoter, PuroR/NeoR, and BGHpolyA. The expression of selection marker genes, PuroR/NeoR plus BGH polyA is driven by CMV promoter.This donor construct is integrated into the site behind the Hotair promoter.

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H. Cui and Hengmi CuiYangyang Liu, Xiao Han, Junting Yuan, Tuoyu Geng, Shihao Chen, Xuming Hu, Isabelle

Silences Expression of Protein-coding and Non-coding RNA GenesBiallelic Insertion of a Transcriptional Terminator via CRISPR/Cas9 Efficiently

published online February 14, 2017J. Biol. Chem. 

  10.1074/jbc.M116.769034Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2017/02/28/M116.769034.DC1

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