Inhibition of CRISPR/Cas9-Mediated Genome Engineering by a Type I Interferon-Induced Reduction in Guide RNA Expression

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome engineering technology is a powerful tool for generation of cells and animals with engineered mutations in their genomes. In order to introduce the CRISPR/Cas9 system into target cells, nonviral and viral vectors are often used; however, such vectors trigger innate immune responses associated with production of type I interferons (IFNs). We have recently demonstrated that type I IFNs inhibit short-hairpin RNA-mediated gene silencing, which led us to hypothesize that type I IFNs may also inhibit CRISPR/Cas9-mediated genome mutagenesis. Here we investigated this hypothesis. A single-strand annealing assay using a reporter plasmid demonstrated that CRISPR/Cas9-mediated cleavage efficiencies of the target double-stranded DNA were significantly reduced by IFNα. A mismatch recognition nuclease-dependent genotyping assay also demonstrated that IFNα reduced insertion or deletion (indel) mutation levels by approximately half. Treatment with IFNα did not alter Cas9 protein expression levels, whereas the copy numbers of guide RNA (gRNA) were significantly reduced by IFNα stimulation. These results indicate that type I IFNs significantly reduce gRNA expression levels following introduction of the CRISPR/Cas9 system in the cells, leading to a reduction in the efficiencies of CRISPR/Cas9-mediated genome mutagenesis. Our findings provide important clues for the achievement of efficient genome engineering using the CRISPR/Cas9 system.

Genome engineering technologies are powerful tools and are widely used for studying the functions of genome sequences.¹⁾ In order to engineer target genomic DNA, artificial nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are introduced into target cells. These nucleases recognize the specific target DNA via their DNA-binding domain in a sequence-specific manner, digest the target genomic DNA, and produce double-stranded DNA breaks (DSBs). The resulting DSBs are subjected to error-prone non-homologous end joining (NHEJ), leading to introduction of insertion or deletion (indel) mutations into the target genomic DNA.¹⁾ In addition to the NHEJ, homologous recombination (HR) can be induced in the presence of donor DNA with homology to the target genomic DNA, resulting in the designed mutation.²⁾ However, the design and construction of these artificial nucleases showing efficient cleavage activity require complex procedures and investigators with much prior experience.

Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, which efficiently introduces indel mutations in the target genomic DNA, was developed.^3–5) The CRISPR/Cas9 system was originally found in bacteria and archaea as their adaptive immune system against foreign pathogens including phages and viruses.^6,7) A humanized Cas9 (hCas9) endonuclease and a guide RNA (gRNA), which contains sequences complementary to the target genomic sequences and a hCas9-interaction interface structure, are co-expressed in cells. Subsequently, the gRNA guides the hCas9 endonuclease to the target genomic DNA in a sequence-specific manner, leading to the introduction of DSBs and indel mutations. Since the gRNA expression cassette can be easily designed and constructed using synthetic oligonucleotides, the newly developed CRISPR/Cas9 system was quickly and widely adopted for genome editing.⁵⁾

In order to introduce the CRISPR/Cas9 system into target cells, plasmid vectors are widely used.⁸⁾ In addition, several groups have developed CRISPR/Cas9 system-expressing viral vectors that make it possible to efficiently introduce the CRISPR/Cas9 system in vivo due to their superior transduction properties.^8,9) However, nonviral and viral vectors often trigger innate immune responses associated with type I interferons (IFNs).¹⁰⁾ Recently, we examined the involvement of type I IFN in short-hairpin RNA (shRNA)-mediated knockdown of target genes. Following IFNα stimulation, the processing of shRNA to small-interfering RNA (siRNA) was significantly inhibited in the cells transfected with shRNA-expressing plasmids. shRNA-mediated knockdown of target genes was significantly inhibited by IFNα. These results indicate that type I IFNs inhibit shRNA-mediated RNA interference (RNAi) via inhibition of dicer-mediated processing of shRNA to siRNA.¹¹⁾ These results led us to hypothesize that type I IFNs downregulate not only shRNA-mediated RNAi but also CRISPR/Cas9-mediated genome mutagenesis.

In this study, we examined the efficiencies of CRISPR/Cas9-mediated genome mutagenesis following IFNα stimulation. Treatment with recombinant human IFNα significantly reduced the copy numbers of gRNA in the cells, leading to a reduction in the efficiencies of the CRISPR/Cas9-mediated genome mutagenesis.

MATERIALS AND METHODS

Cells

A549 cells (a human lung adenocarcinoma epithelial cell line) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), streptomycin (100 µg/mL), and penicillin (100 U/mL). H1299 cells (a non-small cell lung carcinoma cell line) were cultured in RPMI1640 supplemented with 10% FBS, streptomycin (100 µg/mL), and penicillin (100 U/mL).

Reagents

Recombinant human IFNα and IFNγ were purchased from PBL Interferon Source (Piscataway, NJ, U.S.A.). Recombinant human tumor necrosis factor (TNF)-α was purchased from Invivogen (San Diego, CA, U.S.A.). Control siRNA (siControl) and siRNA against RNaseL (siRNaseL) were purchased from Qiagen (Allstars Negative Control siRNA; Qiagen, Hilden, Germany) and Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.), respectively.

Plasmids

CRISPR/Cas9 plasmids expressing hCas9 and gRNA that targets the AAVS1 “safe harbor” locus and the enhanced green fluorescent protein (EGFP) gene were constructed by ligating double-stranded oligonucleotides into the BbsI site of pX330 (Addgene plasmid 42230; Addgene, Cambridge, MA, U.S.A.),³⁾ resulting in pX330-gRNA/AAVS1 and -gRNA/EGFP, respectively. The oligonucleotides used in this study are described in Supplementary Table S1. pHMEF-EGFP, the plasmid expressing EGFP, was previously constructed.¹²⁾ pX330-gRNA/Cetn1,¹³⁾ which is a CRISPR/Cas9 plasmid targeting the Centrin 1 (Cetn1), was obtained from Addgene (plasmid 50718) and was used as a negative control.

The pCAG-EGxxFP-AAVS1 validation plasmid, which was used to validate the DSB production efficiencies of CRISPR/Cas9 plasmids (see also Fig. 1B), was constructed as follows. An approximately 500 bp genomic DNA fragment containing the gRNA target sequence in the AAVS1 locus was amplified by PCR, and was inserted into multi-cloning sites between the EGFP fragments in pCAG-EGxxFP¹³⁾ (Addgene plasmid 50716), resulting in pCAG-EGxxFP-AAVS1.

Fig. 1. IFNα-Mediated Inhibition of DSB Production via the CRISPR/Cas9 System

(A) A schematic diagram of pX330-gRNA. U6: a human U6 promoter; gRNA: a guide RNA; CBh: a hybrid chicken β-actin promoter; hCas9: a humanized Cas9 endonuclease; pA: bovine growth hormone (BGH) polyadenylation signal. (B) Reconstitution of the EGFP gene by a homology-dependent DNA repair (HDR). CRIPSR/Cas9-mediated production of double-strand DNA breaks (DSBs) induces HDR in the target sequence of the pCAG-EGxxFP plasmid, resulting in the expression of the EGFP gene. (C–E) H1299 cells were co-transfected with a validation plasmid (pCAG-EGxxFP-AAVS1) and a plasmid carrying the CRISPR/Cas9 system against an AAVS1 locus (px330-gRNA/AAVS1), followed by treatment with recombinant IFNα at 10⁴ U/mL (C, E) or the indicated concentrations (D). After 48-h incubation, phase-contrast and EGFP-fluorescent photomicrographs of the cells were obtained (C, D), and EGFP-positive cell numbers were determined by a flow cytometry (E). The scale bar represents 200 µm.

Cells were then transfected with these plasmids using Lipofectamine 2000 or Lipofectamine 3000 (Life Technologies, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions.

Determination of hCas9 Protein Levels by Western Blotting Analysis

H1299 cells were transfected with the CRISPR/Cas9 plasmid (pX330-gRNA/AAVS1). After 48-h incubation, hCas9 protein levels in the cells were examined by Western blotting analysis. Western blotting analysis was performed as previously described.¹⁴⁾ Briefly, whole-cell extracts were prepared and electrophoresed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels under reducing conditions, followed by electrotransfer to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, U.S.A.). After blocking with 5% skim milk prepared in TBS-T (Tween-20, 0.1%), the membrane was incubated with mouse anti-FLAG (M2) or anti-β-actin (AC-15) antibodies (Sigma-Aldrich Japan, Tokyo, Japan), followed by incubation in the presence of horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse immunoglobulin G (IgG) antibody (Cell Signaling Technology, Danvers, MA, U.S.A.).

Determination of gRNA Copy Numbers by Northern Blotting Analysis

Cells were transfected with the CRISPR/Cas9 plasmid (pX330-gRNA/AAVS1). After 48-h incubation, gRNA copy numbers in the cells were examined by Northern blotting analysis. Northern blotting analysis was performed as previously described.¹⁵⁾ Total RNA was extracted from the cells with ISOGEN (Nippon Gene, Tokyo, Japan). Ten micrograms of total RNA per lane was loaded onto 15% polyacrylamide denaturing gel. After electrophoresis, bands of RNA were transferred to Hybond-N+ membranes (Roche, Mannheim, Germany). The membranes were then probed with ³²P-labeled synthetic oligonucleotides that were complementary to the sequence of gRNA, or human U6 small nuclear RNA (gRNA: 5′-tagcaagttaaaataaggctag-3′; U6: 5′-tgctaatcttctctgtatcgt-3′).

Single Strand Annealing (SSA) Assay

H1299 cells were co-transfected with the validation plasmid (pCAG-EGxxFP-AAVS1) and the CRISPR/Cas9 plasmids (pX330-gRNA/AAVS1 or -gRNA/Cetn1¹³⁾) using Lipofectamine 2000. The EGFP expression levels were evaluated by microscopic observation using a fluorescence microscope and by flowcytometric analysis 48 h after transfection.

Genotyping Assay Using a Mismatch Recognition Nuclease

H1299 cells were transfected with the CRISPR/Cas9 plasmids (pX330-gRNA/AAVS1 or -gRNA/EGFP). After 72-h incubation, the levels of insertion of indel mutations were assessed by a mismatch-dependent nuclease assay using a Guide-it Mutation Detection Kit (Clontech-TaKaRa, Kyoto, Japan). Briefly, genomic DNA of the human AAVS1 locus and the EGFP gene was amplified by PCR using the primers AAVS1-F/R and EGFP-F/R (Supplementary Table S1) and the total DNA of the cells transfected with CRISPR/Cas9 plasmids. The resulting PCR amplicons were then denatured and reannealed by heating and gradual cooling. The DNA fragments with potential mismatch mutations were digested by a mismatch-sensitive enzyme, Guide-it Resolvase, followed by a gel-electrophoresis analysis. The intensities of each DNA band were quantified using Image J software. The percentages of indel mutations were calculated as previously described.³⁾

Target-Site Genotyping by Sequencing Analysis

Total DNA was isolated from the cells 72 h after co-transfection with pX330-gRNA/EGFP and pHMEF-EGFP. The target region in the EGFP-expressing plasmid was amplified by PCR using the primers EGFP-F/R (Supplementary Table S1). The resulting PCR amplicons were then cloned to a plasmid for sequencing using a TOPO TA Cloning Kit (Life Technologies). Sequencing of the cloned DNA was performed at Fasmac (Kanagawa, Japan).

RESULTS

In order to examine whether the type I IFNs affect the cleavage efficiencies of the CRISPR/Cas9 system, H1299 cells were transfected with a CRISPR/Cas9 plasmid targeting the human AAVS1 locus (pX330-gRNA/AAVS1) (Fig. 1A), followed by treatment with recombinant human IFNα. Quantitative RT-PCR analysis showed that IFNα treatment induced a significant increase in the expression of IFN-stimulated genes (ISGs) (Supplementary Fig. S1), indicating that H1299 cells were type I IFN-responsive. Transfection efficiencies, which were assessed by the percentages of EGFP-positive cells following transfection with an EGFP-expressing plasmid (pHMEF-EGFP), in H1299 cells were not significantly altered by treatment with recombinant human IFNα 48 h after transfection (Supplementary Fig. S2). First, we examined the efficiencies of CRISPR/Cas9-mediated DSBs by an SSA assay^13,16,17) using a reporter plasmid (pCAG-EGxxFP-AAVS1) (Fig. 1B). The plasmid shows EGFP expression, when the EGFP expression cassette is reconstituted by homology-dependent repair (HDR) following CRISPR/Cas9-mediated DSBs at the AAVS1 genomic DNA cloned into pCAG-EGxxFP (Fig. 1C). In the absence of IFNα, significant EGFP expression was observed following co-transfection with pX330-AAVS1 and pCAG-EGxxFP-AAVS1 (Fig. 1C, left), whereas EGFP expression was significantly reduced in the presence of IFNα (Fig. 1C, right). The reduction in EGFP expression by IFNα was dependent on the concentrations of IFNα (Fig. 1D). Moreover, flow cytometry analysis demonstrated that the percentage of GFP-positive cells was reduced from 15.9 to 9.9% by IFNα (Fig. 1E). EGFP expression was below the level of detectability following transfection with pCAG-EFxxFP-AAVS1 alone or co-transfection with pCAG-EFxxFP-AAVS1 and pX330-gRNA/Cetn1, which is a CRISPR/Cas9 plasmid targeting Cetn1 gene and was used here as a negative control (Supplementary Fig. S3). These results suggest that IFNα reduces the DSB efficiencies of the CRISPR/Cas9 system.

Next, a genotyping assay^3,18) of the AAVS1 genomic DNA using a mismatch recognition nuclease was performed. In this assay, the target DNA sequence was amplified by PCR. Subsequently, the PCR products were denatured and reannealed, followed by digestion with a mismatch-dependent nuclease. The cleavage products were not significantly detected in mock-transfected cells (Fig. 2). On the other hand, the apparent cleavage products were found in the cells transfected with pX330-gRNA/AAVS1 without IFNα (Fig. 2). Densitometric analysis of the PCR product bands on the gels demonstrated that the percentage of indel mutations was 9%. On the other hand, IFNα stimulation reduced the percentage of indel mutations to 5%. These results indicate that IFNα mediates the reduction in the efficiencies of target genome mutagenesis via the CRISPR/Cas9 system. In order to further examine the effects of IFNα on CRISPR/Cas9-mediated genome mutagenesis, we constructed another CRISPR/Cas9 plasmid targeting the EGFP gene (pX330-gRNA/EGFP). H1299 cells were co-transfected with pX330-gRNA/EGFP and an EGFP-expressing plasmid (pHMEF-EGFP), followed by treatment with recombinant human IFNα. No significant level of cleaved product was detected after transfection with pHMEF-EGFP alone or co-transfection with pX330-gRNA/Cetn1 and pHMEF-EGFP, while the apparent cleaved products were detected in the cells co-transfected with pX330-gRNA/EGFP and pHMEF-EGFP in the absence of IFNα (Fig. 3A). Densitometric analysis demonstrated that the insertion of indel mutations was reduced from 17 to 8% by IFNα treatment (Fig. 3A). Finally, the target genome region was amplified by PCR, and then the sequence of the target region was directly analyzed. In the absence of IFNα, 33.3% of the analyzed target genome sequences carried the mutations (Fig. 3B). On the other hand, the mutation frequency was reduced to 11.1% by IFNα stimulation (Fig. 3B). These results indicate that type I IFNs inhibit CRISPR/Cas9-meditaed genome mutagenesis.

Fig. 2. IFNα-Mediated Inhibition of Genome Mutagenesis via the CRISPR/Cas9 System

H1299 cells were transfected with px330-gRNA/AAVS1, followed by treatment with recombinant IFNα at 10⁴ U/mL. After 72-h incubation, genome mutagenesis efficiencies in the cells were assessed by a genotyping assay using mismatch recognition nuclease. Band intensities of the cleaved PCR products were quantified using Image J software.

Fig. 3. IFNα-Mediated Inhibition of Genome Mutagenesis Following Transfection with pX330-gRNA/EGFP

H1299 cells were co-transfected with a plasmid expressing EGFP (pHMEF-EGFP) and a plasmid carrying the CRISPR/Cas9 system against an EGFP gene locus (px330-gRNA/EGFP), followed by treatment with recombinant IFNα at 10⁴ U/mL. (A) After 72-h incubation, genome mutagenesis efficiencies in the cells were assessed by a genotyping assay using a mismatch recognition nuclease. Band intensities of the cleaved PCR products were quantified using Image J software. (B) After 72-h incubation, genomic DNA in the cells was extracted, and frequencies of mutation were quantified by sequencing analysis. The numbers of mutated samples are indicated in parenthesis (mutated/analyzed samples).

Finally, we examined the effects of type I IFNs on hCas9 and gRNA expression levels in the cells transfected with pX330-gRNA/AAVS1. The protein levels of hCas9 were not significantly altered by IFNα in H1299 cells (Fig. 4A). On the other hand, Northern blotting analysis demonstrated a significant decrease in the copy numbers of gRNA following IFNα stimulation in H1299 and A549 cells (Fig. 4B). In order to examine the effects of other cytokines on gRNA expression levels, H1299 cells were transfected with pX330-gRNA/AAVS1, followed by treatment with the type II IFN (IFNγ) or TNF-α, which is a representative inflammatory cytokine. IFNγ treatment did not alter the gRNA copy numbers, but the copy numbers of gRNA were clearly reduced by TNFα (Fig. 4C). These results indicate that type I IFNs and TNFα, but not type II IFN, reduce the copy numbers of gRNA in the cells, leading to a reduction in the efficiencies of the CRISPR/Cas9-mediated genome mutagenesis.

Fig. 4. IFNα-Mediated Reduction in the Copy Numbers of gRNA Following Transfection with CRISPR/Cas9 Plasmids

H1299 (A, B) and A549 (B) cells were transfected with pX330-gRNA/AAVS1, followed by treatment with recombinant IFNα at 10⁴ U/mL. After 48-h incubation, hCas9 protein expression (A) and gRNA copy numbers (B) in the cells were quantified by Western and Northern blotting analysis, respectively. (C) H1299 cells were transfected with pX330-gRNA/AAVS1, followed by treatment with recombinant IFNγ or TNFα at 10⁴ U/mL. After 48-h incubation, gRNA copy numbers in the cells were quantified by Northern blotting analysis.

IFNα treatment quickly induces ISG expression. Some ISGs might mediate the degradation of gRNA. Double stranded RNA (dsRNA) activates 2′,5′-oligoadenylate synthase (OAS), resulting in production of 2′,5′-oligoadenylates.¹⁹⁾ RNaseL, which can degrade foreign RNAs, is activated by 2′,5′-oligoadenylates. Type I IFN-mediated activation of OAS/RNaseL might induce the degradation of gRNA. In order to examine the effects of OAS/RNaseL pathway on the IFNα-mediated reduction in gRNA copy numbers, H1299 cells were transfected with siRNaseL, followed by transfection with pX330-gRNA/AAVS1 and treatment with IFNα. Quantitative RT-PCR analysis demonstrated that RNaseL was sufficiently knocked down following transfection with siRNaseL (Supplementary Fig. S4A). The copy numbers of gRNA were significantly reduced by IFNα even in RNaseL-knockdown cells as well as siControl-transfected cells (Supplementary Fig. S4B). These results suggest that ISGs other than OAS/RNaseL might be involved in the IFNα-mediated reduction in gRNA copy numbers.

DISCUSSION

Genome engineering technologies using artificial nucleases, including ZFNs and TALENs, have been demonstrated to efficiently introduce designed mutations into various types of cultured cells and animals¹⁾; however, ZFNs and TALENs have not been widely used because of the difficulties in the design and construction of these artificial nucleases. On the other hand, the CRISPR/Cas9 system is already in widespread use due to several advantages, including its superior genome editing efficiencies and ease of design and construction. Numerous studies of CRISPR/Cas9-mediated genome engineering have recently been reported.^3–5,13) On the other hand, the factors affecting the efficiencies of CRISPR/Cas9-mediated genome mutagenesis remain to be evaluated. In this study, we demonstrated that type I IFNs mediate a reduction in gRNA expression levels following transfection with CRISPR/Cas9 plasmids (Fig. 4B), leading to a reduction in the efficiencies of CRISPR/Cas9-mediated genome mutagenesis (Figs. 1–3). These results indicate that we should pay attention to the production of type I IFNs associated with introduction of the CRISPR/Cas9 system.

As shown in Fig. 4B, the copy numbers of the gRNA, which was driven by a U6 promoter, were reduced by type I IFNs in the CRISPR/Cas9 plasmid-transfected cells, whereas the copy numbers of the endogenous U6 RNA were not. We recently demonstrated that type I IFN inhibited the processing, but not transcription, of shRNA.¹¹⁾ These findings suggest that the transcriptional levels of gRNA were not altered by type I IFN treatment. Although it remains unclear why type I IFNs reduce the copy numbers of gRNA, type I IFN-induced expression of ISGs might mediate the degradation of gRNA. RNaseL is a representative ISG with RNA cleavage activity and is activated following OAS-mediated production of 2′,5′-oligoadenylates.¹⁹⁾ However, siRNA-mediated RNaseL knockdown did not restore the gRNA copy numbers in the IFNα-treated cells (Supplementary Fig. S4). Further studies will be necessary to identify cellular factors involved in the type I IFN-mediated reduction of gRNA copy numbers.

In addition of the reduction of the gRNA copy numbers, other mechanisms might be involved in the type I IFN-mediated suppression of the CRISPR/Cas9-mediated genome mutagenesis. Indels are introduced by NHEJ following insertion of DSBs in the CRISPR/Cas9 system.¹⁾ NHEJ is mediated by various proteins, including the DNA-dependent protein kinase (DNA-PK) and Ku70-Ku80 dimer.²⁰⁾ The expression levels or functions of proteins associated with NHEJ might be altered by type I IFNs. Further studies will be needed to elucidate the mechanism underlying the inhibition of CRISPR/Cas9-mediated genome mutagenesis by type I IFNs.

Various groups have reported that non-viral and viral vectors carrying the CRISPR/Cas9 system are promising therapeutic agents for severe diseases, because the CRISPR/Cas9 system is able to mediate correction of the genome locus coding the disease-related gene in animal models.^5,21) In addition, a CRISPR/Cas9 system targeting the hepatitis B virus (HBV) genome efficiently inhibited the viral replication.^22,23) In particular, the CRISPR/Cas9 system is promising for cleavage of HBV covalently closed circular DNA (cccDNA), which plays a crucial role in persistent HBV infection and is an important target for anti-HBV drugs; however, there are no drugs which can eliminate cccDNA from the cells. In the future, we should pay close attention to the application of CRISPR/Cas9 systems to HBV treatment. Treatment with pegylated IFNα (PEG-IFN) is often given to patients with persistent HBV infection.^24,25) Combined therapies with PEG-IFN and HBV-targeting CRISPR/Cas9 might exhibit reduced therapeutic efficacy, because PEG-IFN would be expected to inhibit CRISPR/Cas9-mediated mutagenesis activity in the HBV genome.

In summary, we have demonstrated that type I IFNs inhibit CRISPR/Cas9-mediated genome mutagenesis via a reduction in gRNA expression. This study strongly suggests that it is important to avoid IFN responses for efficient engineering of the target genome locus by the CRISPR/Cas9 system.

Acknowledgments

We thank Sayuri Okamoto and Eri Hosoyamada (Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan) for their help. We thank Marcos Andrés Taracena (Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan) for critical reading of the manuscript. We also thank Masahito Ikawa for his help (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan). This work was supported by Grants-in-Aid for Scientific Research (A) [26242048] and (B) [26293118] from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and by a Grant-in-Aid from the Ministry of Health, Labour, and Welfare (MHLW) of Japan and Japan Agency for Medical Research and Development (AMED) [15fk0310017h0004]. M. Machitani and K. Wakabayashi are Research Fellows of the Japan Society for the Promotion of Science.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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