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). efficiently silenced the transcription of its target gene Taken together, these findings indicated that even if the integration took place in the intron of the the BGH PolyA signal driven by an endogenous pro- gene. moter was able to efficiently terminate its down- Efficient selection for biallelic silencing Downloaded from stream transcription. Because BGH PolyA is a short by double selection markers. A single marker terminator, its integration into a target gene may selection for PolyA signal integration was not ef- be more efficiently mediated by a CRISPR/Cas9- ficient and only 4.9% of the clonal cell lines showed

induced HDR approach than by other long termina- biallelic integration of the PolyA signal into the http://www.jbc.org/ tors (i.e., 4× BGH PolyA signals and the beta-globin PPP1R12C gene (Table 1). This low efficiency terminator). We thus speculated that a biallelic may become worse if genes do not respond well BGH PolyA integration mediated by CRISPR/Cas9- to Cas9/gRNA or HDR (11). We therefore tested induced HDR could efficiently shut down transcrip- whether double selection markers (e.g., neomycin tion of a target gene. resistance (NeoR) and PuroR for drug selection) by guest on May 8, 2020 The transcription of the PPP1R12C gene could improve the efficiency of the biallelic silenc- was terminated by an integrated BGH PolyA sig- ing. The plasmid with NeoR-BGH PolyA cassette nal. To silence the PPP1R12C gene, a protein- was constructed in the same way as the plasmid coding gene, by biallelic integration of a PolyA sig- with PuroR-BGH PolyA cassette (Figure 4A). The nal via CRISPR/Cas9-induced HDR, a donor plas- two plasmids were co-transfected into HEK293 cells mid was constructed for integrating a PolyA sig- with the Cas9/AAVS1-gRNA plasmids. Genotyping nal into the adeno-associated virus integration site 1 analysis showed that, after two weeks of double drug (AAVS1) in the first intron of the PPP1R12C gene selection, 94.2% of clonal cell lines were bialleli- based on gene trapping strategy (Figure 3A). AAVS1 cally transgenic, and only 4 out of 69 (5.8%) clonal is a “safe harbor” gene located within the first in- cell lines were monoallelically transgenic ((Figure tron of the human PPP1R12C gene citemali2013rna. 4C), Table 1). The biallelic integration of the PolyA This plasmid contains a splice adopter (SA), T2A, a signal was mediated by Cas9/gRNA-induced HDR puromycin resistance (PuroR) selection marker and (Figure 4B). Moreover, qRT-PCR analysis on the a BGH PolyA signal, flanked by homologous se- clonal lines revealed that the transcription of the quences of AAVS1 gene (Figure 3B). The donor PPP1R12C gene was dramatically shut down (Fig- plasmid was co-transfected into HEK293 cells with ure 4D). Together, these findings indicated that bial- the plasmids expressing the Cas9 protein and the lelic silencing was largely improved by the double AAVS1 guide RNA (gRNA). A PCR-based geno- marker selection. typing analysis using genomic DNA revealed that Silencing the lncRNA gene by biallelic in- 93.5% of clonal cell lines were transgenic after 2 tegration of a PolyA signal. MALAT1 is a long weeks of puromycin selection, with only 3 out of noncoding RNA (lncRNA) gene that is highly ex- 62 (4.9%) clonal lines having biallelic transgenes pressed in a variety of cell lines (e.g., A549, Hela,

3 gene silencing using PolyA signal via CRISPR/Cas9 system and HepG2). Because silencing lncRNA is consid- gration of a PolyA signal into the first exon of the ered a challenge, we tested whether the approach NSUN2 gene efficiently terminated the transcription used to silence the PPP1R12C gene as described of this protein-coding gene. above could also be applied to lncRNA genes. For this purpose, two donor plasmids were constructed DISCUSSION containing a 5’-homologous arm (HA), PuroR (or Gene deletion or knockout is a useful tool NeoR), the BGH PolyA signal, and a 3’-HA (Figure for gene functional studies. However, it is often nec- 5A). A plasmid containing gRNA targeting to the se- essary to remove the whole sequence of the targeted quences immediately behind the MALAT1 promoter gene or a large DNA fragment surrounding the pro- was also constructed. These donor plasmids did not moter or the whole gene (8, 11). This can cause include SA-T2A sequences as MALAT1 is a non- a misinterpretation of the function of the targeted protein- coding gene. These plasmids, together with gene because it is possible that a gene on the com- the Cas9-containing plasmid, were co-transfected plimentary DNA strand overlaps with the targeted into HepG2 cells. After two weeks of double drug gene and/or its regulatory sequence or that a partial selection, 37 out of 47 (78.7%) clonal cell lines were coding sequence is also removed due to this over- biallelically transgenic with the insertion of NeoR lap (e.g., the genomic region that can be transcribed (or PuroR) and the BGH PolyA signal sequences (Ta- into sense and antisense transcripts). RNA interfer- Downloaded from ble1), as indicated by the genotyping analysis using ence is another useful tool commonly used for gene specific primers (MALAT1-HDR-F/R) for the trans- silencing, but it also has several shortcomings, in- genic fragments (Figure 5B). The qRT-PCR analysis cluding a high off-targeting rate and insufficient gene

showed that the mRNA abundance of MALAT1 in silencing (16). http://www.jbc.org/ the cells with biallelic integration of the PolyA signal In this study, we developed a novel approach was reduced to 0.1% compared to the control cells to gene silencing using the biallelic integration of (Figure 5C). These findings suggested that biallelic a PolyA signal mediated by CRISPR/Cas9-induced integration of the PolyA signal via CRISPR/Cas9- HDR. The CRISPR/Cas9 system is a tool for gene induced HDR efficiently silenced the transcription editing that has been recently developed for gene by guest on May 8, 2020 of a long non-protein-coding gene, MALAT1. knockout and knock-in (5, 17) . Compared to other Silencing NSUN2 by biallelic integration gene editing tools, it is simple, easy and convenient of a PolyA signal. We also tested whether biallelic and has the potential to target multiple genes si- integration of a PolyA signal into the open reading multaneously. More importantly, the CRISPR/Cas9 frame of the NSUN2 gene, a RNA methyltransferse system can significantly increase the rate of HDR- gene, could disrupt the transcription of this protein- mediated biallelic integration (5, 18). In this study, coding gene. Similarly, two donor plasmids were we leveraged the power of the CRISPR/Cas9 system constructed containing a 5’-HA, T2A, PuroR (or and used a BGH PolyA signal together with two se- NeoR), the BGH PolyA signal, and a 3’-HA (Figure lection markers to successfully integrate constructs 6A). The plasmid containing gRNA targeting to the into PPP1R12C, NSUN2, and MALAT1 at three dif- first exon of the NSUN2 gene was also constructed. ferent sites (i.e., the first intron of PPP1R12C, the These plasmids, together with the Cas9-containing first exon of NSUN2, and directly behind the pro- plasmid, were co-transfected into HEK293 cells. moter of MALAT1). Because this approach does After two weeks of double drug selection, 17 out of not involve either the removal of large DNA frag- 26 (65.4%) clonal cell lines were biallelically trans- ments or RNA interference, it overcomes some of genic (Table 1), as indicated by genotyping anal- the shortcomings of the conventional gene editing ysis using specific primers (NSUN2-HDR-F/R) for tools such as the low efficiency of HDR-mediated the transgenic fragments (Figure 6B). The qRT-PCR biallelic integration and the extensive screening of analysis showed the expression of NSUN2 in the clonal cells (4, 18). In addition, the ability to choose cells with biallelic integration of PolyA signal was from multiple integration sites allows the side ef- reduced to 1-3% compared to the control cells (Fig- fects of the integrated PolyA signal in the targeted ure 6C). These results suggested that biallelic inte- or overlapping genes to be prevented. Moreover,

4 gene silencing using PolyA signal via CRISPR/Cas9 system as indicated by qRT-PCR analysis, gene silencing for PolyA integration sites to achieve the best si- by biallelic integration of a PolyA signal did not lencing efficiency based on the specificity of a target completely block the transcription of the targeted gene. Using this approach, we can establish a cell gene. This feature makes the novel approach espe- line with a coding or non-coding gene silencing very cially useful for functional studies of genes whose efficiently and shortly. deletions are lethal. The successful integration of the BGH EXPERIMENTAL PROCEDURES PolyA signal significantly shut down the transcrip- Cell Culture and Transfection. The hu- tion of three genes, including two protein-coding man embryonic kidney cell line 293 (HEK293) was genes (PPP1R12C and NSUN2) and a long non- purchased from ATCC (CRL-1573, USA). Cells protein-coding gene, MALAT1. Although it is not were maintained in Dulbecco’s modified Eagle’s surprising that the protein-coding genes were si- medium (DMEM) supplemented with 10% fetal lenced through the novel approach, the almost com- bovine serum (HyClone, USA), 2 mM GlutaMAX plete shutdown of the long non protein-coding gene (Life Technologies, USA), 100 U/ml penicillin and implies that this approach can be applied for silenc- 100 mg/ml streptomycin at 37°C with 5% CO2. For ing almost any gene of interest. transfection, cells were seeded into 60 mm dishes In this study, the efficiency of gene silenc- (Corning, USA) at a density of 2×106 cells/dish Downloaded from ing by biallelic integration of a PolyA signal was and cultured in an antibiotic-free medium. When greatly improved by double marker selection. Al- cells were at 80%–90% confluency, they were trans- though the marker genes were driven by the endoge- fected with plasmids using Lipofectamine 2000 (Life

nous promoter of the targeted gene, expression of Technologies, USA) according to the manufacturer’s http://www.jbc.org/ the marker genes appeared to be sufficient for effec- instructions. For PolyA testing, cells were trans- tive selection. The results from the single marker fected with a plasmid mixture (a total 4 µg of BGH- selection study showed that only 4.8% of clonal cell puroR and BGH-neoR donor plasmids and 4 µg of lines were biallelically transgenic. In contrast, the Cas9/sgRNA plasmid). After 2 days of transfec- double marker selection lead to 65-93% of biallelic tion, cells were treated with 1 µg/mL of Puromycin by guest on May 8, 2020 integration by two-week selection. Based on these (Sigma, USA) for 3 days, and then followed by treat- results, we can speculate that double marker selec- ment with 400 µg/ml of Neomycin (Sigma, USA) for tion may be particularly useful for the cell lines with 14 days. low biallelic integration efficiency. If we could pro- Construction of PolyA-Contained Plas- long antibiotics selection time (e.g. from two weeks mids. The BGH PolyA signal was amplified from to three weeks, we would have much better biallelic a pcDNA3.1 (+) vector by PCR. The 4 × PolyA transgenic rates. Nevertheless, if the endogenous was constructed with 4 PolyAs using a Golden gate promoter of the targeted gene was very weak, this clone. The beta-globin PolyA was also amplified approach would not be appropriate because the ex- from HEK293 cell genomic DNA following previ- pression of marker genes may not be sufficient for ous published protocols (14). Four different PolyA drug selection. For example, the expression of the signals were inserted into the EcoRI site of pIRES2- HOTAIR gene in HEK293 cells was very low (Fig- EGFP (Clontech, USA), followed by the replacement ure7)and under this promoter, the integrated marker of the CMV promoter with the EF-1A promoter. gene had a very low expression, leading to the failure The four vectors containing the viral CMV promot- of drug selection (data not shown). In this case, an ers were digested with BglII and ligated with the additional promoter (e.g., EF-1A) may be inserted RFP gene derived from pGBT-RP2-1. A list of all in front of the marker gene to drive its expression cloning primers are listed in the Supplemental Table (Figure8)(12). In conclusion, we tried three dif- S1. ferent insertion targets for PolyA integration, i.e., an Construction of vectors. The sgRNA se- integration site within the first intron, within the first quences were synthesized and annealed to the Cas9 exon, or immediately behind the promoter of a tar- expression plasmid (Addgene ID 42230, USA) ac- get gene. These strategies provide multiple choices cording to a previously published protocol (19). The

5 gene silencing using PolyA signal via CRISPR/Cas9 system

Neo-BGH poly (A) donor was constructed as previ- 5 min to inactivate the proteinase K. Five ml sample ously described (20, 21). In brief, the NeoR and were used for a subsequent integration-oriented PCR PuroR genes were subcloned from the pcDNA3.1 with 2 × Taq Master Mixture (Vazyme, city, country) (+) vector (Invitrogen) and the pSMPUW-IRES- according to the manufacturer’s recommendations. Puro vector (Cell Biolabs, USA), respectively. The Luciferase Reporter Assays for Promoter NeoR and PuroR genes were then linked with BGH Activity. The reporter vector of pGL3 (Promega, PolyA signals through overlapping PCR. The left city, country) was used for transfection into HEK293 and right homology sequences were cloned from cells. We first generated several promoter sequences HEK293 cell genomic DNA. The pGEM®-T Easy by PCR with the primers listed in Table S1. The frag- Vector (Promega, city, country) was amplified with ments included the MALAT1 large promoter (nu- a pair of oligo primers containing the BsmbI re- cleotides +45 to -1047bp, of the TSS), the HDR- striction site and used as a vector backbone. To MALAT1 promoter (nucleotides +45 to -729bp), generate NeoR/PuroR-BGH poly (A) donor vec- and the HOTAIR promoter (nucleotides +204 to tors, NeoR/PuroR-BGH poly (A) and homology -904bp of the TSS in NR047518.1). These pro- sequences generated by amplification with BbsI moter fragments were respectively cloned into the site-containing oligo primers were cloned into the pGL3-promoter vector using two cloning sites (XhoI BbsI-digested vector using the Golden Gate cloning and HindIII). The constructs were named MALAT1- Downloaded from method. large, MALAT1-small, HDR-MALAT1 and pGL3- Reverse transcription and qPCR. Cells Hotair, respectively. HEK293 cells were seeded at were disrupted in TRIzol (Life Tech, city, country) a density of 4 × 105 cells per well in 12-well plates

and RNA was extracted according to the manufac- 24 hours before transfection. Four hundred ng lu- http://www.jbc.org/ turer’s instructions. Complementary DNAs were ciferase plasmid containing different promoters were synthesized using the PrimeScriptTM RT reagent co-transfected with 2 ng of pRL-TK plasmid. The Kit plus gDNA Eraser (Takara, city, country) and PGL3-control and PGL3-Basic constructs were used random primers. Quantitative PCR was performed as positive and negative controls, respectively. The using SYBR Premix Ex Taq (Takara, DDR420A, Lipofectamine® 2000 transfection reagent (Invitro- by guest on May 8, 2020 city, country). Regular PCR was performed using gen, USA) was used. Forty-eight hours after trans- Taq polymerase (Fermentas, city, country) following fection, the luciferase activity was measured and nor- manufacturer’s recommendations. malized. Isolation of genomic DNA and PCR- FACS analysis. HEK293 cells were trans- based genotyping analysis. Cells in 96-well plates fected with the plasmid containing the EGFP and were lysed with 0.1 ml lysis buffer containing 10 RFP genes. Cells were then analyzed on a FACS mM Tris pH 8, 2 mM EDTA, 0.2% Triton X-100, Aria II cell sorter (BD Biosciences, USA) after 24 and 200 µg/mL Proteinase K. After 2 hours of incu- hours of incubation. All primer sequence informa- bating at 50-56°C, the cells were heated at 95°C for tion in the experiment can be obtained by inquiry.

Acknowledgments: This research was supported by the National Key Research and Development Program in China (2016YFC1303604), the National Natural Science Foundation of China (81171965, 81372237 and 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 this article.

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. All authors read and approved the final manuscript.

6 gene silencing using PolyA signal via CRISPR/Cas9 system

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8 gene silencing using PolyA signal via CRISPR/Cas9 system

FOOTNOTES 5’HA: 5’ left arm homologous, CMV promoter: cytomegalovirus enhancer and myeloproliferative sarcoma virus promoter, PuroR: puromycin resistancegene, NeoR: neomycin resistancegene, BGH polyA: bovine growth hormone polyadenylation, 3’HA : 3’ right arm homologous.

TABLES

Silenced gene Integration site Selection marker Total No HDR Homozygous Heterozygous All HDR PPP1R12C 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 Downloaded from http://www.jbc.org/ by guest on May 8, 2020

9 gene silencing using PolyA signal via CRISPR/Cas9 system

FIGURES Downloaded from http://www.jbc.org/

Figure 1: Analysis of silencing potency for different silencing signals driven by viral promoter.(A) The structures of different plasmids constructed for this analysis. The control plasmid is highlighted with a viral promoter, CMV promoter (blue arrow), RFP (red box), IRES (orange box) and EGFP (green box).The other plasmids are constructed from control plasmid. The difference of the plasmids from control plasmid is the by guest on May 8, 2020 different silencing signals inserted between RFP and IRES, including BGH PolyA(+) (black arrow),4xBGH PolyA (4 black arrows), and beta-globin terminator (long black arrow).

10 gene silencing using PolyA signal via CRISPR/Cas9 system Downloaded from http://www.jbc.org/

Figure 2: Analysis of silencing potency for different terminator signals driven by an endogenous EF-1A promoter.(A) The structures of plasmids with different terminator signals. The control plasmid consists of an endogenous promoter, EF-1A promoter (blue arrow), IRES (orange box) and EGFP (green box) without terminator signal. The other plasmids are constructed based on the control plasmid with the by guest on May 8, 2020 different terminator signals inserted between EF-1A promoter and IRES, including BGH polyA(+) (a black arrow), 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 with different plasmids containing different terminator signals. Compared to control plasmid, the other plasmids efficiently silenced the transcription except for BGH PolyA(-) plasmid. The transcription is presented as fold changes 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 PolyA sequences; IRES, internal ribosomal entry site sequences, EGFP: enhanced green fluorescent protein gene.

11 gene silencing using PolyA signal via CRISPR/Cas9 system Downloaded from http://www.jbc.org/

Figure 3: Crispr/Cas9-based HDR-mediated biallelic integration of polyA into the first intron of

PPP1R12C or the site of AAVS1 causes downstream transcriptional termination.(A) A schematic by guest on May 8, 2020 overview 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 lines with wild type of PPP1R12C, or with monoallelic/biallelic integration of polyA signal into the first intron of PPP1R12C 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 the bottom showing wild type is homozygous for PPP1R12C gene as indicated by 1976bp single amplicon, clonal cell line 17 and 23 are both homozygous for biallelically integrated polyA signals as indicated by 3310bp single 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, and 3/4Exon-F/R, a set for exon 3 and 4 of PPP1R12C) indicates that, compared to HEK293 cells transfected with control plasmid, the abundance of the targeted transcripts is drastically reduced. GAPDH is used as internal control. N=3. The data are presented as the means + standard deviation (SD).

12 gene silencing using PolyA signal via CRISPR/Cas9 system Downloaded from

Figure 4: Crispr/Cas9-based gene silencing by biallelic integration of polyA signals is efficiently im- http://www.jbc.org/ proved with the use of double selection markers.(A) A schematic overview of donor plasmids constructed for double drug selection. (B) PCR-based genotyping analysis for identifying clonal cell lines with wild type of PPP1R12C, or with biallelic integration of polyA signal into the first intron of PPP1R12C 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). (C) The gel image wild type is homozygous for by guest on May 8, 2020 PPP1R12C gene as indicated by 1976bp single amplicon, clonal cell lines 6, 8, 15 and 16 are all homozygous for 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 to HepG2 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).

13 gene silencing using PolyA signal via CRISPR/Cas9 system Downloaded from

Figure 5: Crispr/Cas9-based HDR-mediated biallelic integration of polyA into the site just behind the promoter of MALAT1 causes downstream transcriptional termination.(A) Schematic diagram il- lustrating the strategy for MALAT1 gene silencing through homology directed repair (HDR) approach. The donor plasmids contain left and right arms (5’-and 3’-HA) homologous to MALAT1, PuroR/NeoR, and http://www.jbc.org/ BGH polyA. The region in yellow denotes promoter, which partially overlaps with 5’-HA. 5’HA: 5’ left arm homologous to MALAT1, PuroR: puromycin resistancegene, NeoR: neomycin resistancegene, BGH polyA: bovine growth hormone polyadenylation, 3’HA : 3’ right arm homologous to MALAT1. (B) PCR-based genotyping analysis for identifying clonal cell lines with wild type of MALAT1, or with biallelic integration of polyA signal into the site just behind the promoter of MALAT1 gene. Primers used for genotyping analysis by guest on May 8, 2020 are 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 1831bp single amplicon, clonal cell lines 2, 3, 5 and 7 are all homozygous for biallelically integrated polyA signals as 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 the targeted transcript is drastically reduced. GAPDH is used as internal control. N=4. The data are presented as the means + standard deviation (SD).

14 gene silencing using PolyA signal via CRISPR/Cas9 system Downloaded from

Figure 6: Crispr/Cas9-based HDR-mediated biallelic integration of polyA into the first exon of NSUN2 causes downstream transcriptional termination.(A) Schematic diagram illustrating the strat- egy for NSUN2 gene silencing through homology directed repair (HDR) approach. The donor plasmids http://www.jbc.org/ contain left and right arms (5’-and 3’-HA) homologous to NSUN2 exon1, T2A, PuroR/NeoR, and BGH polyA. The sequence of gRNA (in red) against NSUN2 is shown. 5’HA: 5’ left arm homologous to NSUN2 exon1, 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) by guest on May 8, 2020 PCR-based genotyping analysis for identifying clonal cell lines with wild type of NSUN2, or with biallelic integration of polyA signal into the first exon of NSUN2 gene. Primers used for genotyping analysis are named as NSUN2-HDR-F/R, which specifically bind to NSUN2 at left and right homologous arms (5’-and 3’-HA). The gel image showing wild type is homozygous for NSUN2 gene as indicated by 1981bp single amplicon, 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 targeted transcript is drastically reduced. GAPDH is used as internal control. N=4. The data are presented as the means + standard deviation (SD).

15 gene silencing using PolyA signal via CRISPR/Cas9 system Downloaded from

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-control plasmid, which contains a control promoter sequence, is used as a positive control. The other plasmids, containing MALAT1 promoter, HDR-MALAT1 promoter, PPP1R12C promoter, or Hotair promoter, are con- http://www.jbc.org/ structed from pGL3-control. HDR sequence in pGL3-HDR-MALAT1 denotes the sequence homologous to MALAT1 is constructed into plasmid for homology-directed repair (HDR). by guest on May 8, 2020

16 gene silencing using PolyA signal via CRISPR/Cas9 system

Figure 8: Schematic illustration of the Crispr/Cas9-based strategy for Hotair gene silencing by biallelic integration of polyA signal through homology directed repair (HDR) approach.The donor plasmid contains left and right arms (5’-and 3’-HA) homologous to Hotair, CMV promoter, PuroR/NeoR, and BGH polyA. 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. Downloaded from http://www.jbc.org/ by guest on May 8, 2020

17 Biallelic Insertion of a Transcriptional Terminator via CRISPR/Cas9 Efficiently Silences Expression of Protein-coding and Non-coding RNA Genes Yangyang Liu, Xiao Han, Junting Yuan, Tuoyu Geng, Shihao Chen, Xuming Hu, Isabelle H. Cui and Hengmi Cui J. Biol. Chem. published online February 14, 2017

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