CRISPR/Cas9-Mediated Disruption of ZNF543 Gene: An Approach Toward Discovering Its Relation to TRIM28 Gene in Parkinson’s Disease

Genetic studies of familial forms of Parkinson’s disease (PD) have shown that the ZNF543 gene is a candidate gene that operates relevant to this disease. However, until now, there is no evidence for ZNF543 gene function in PD, and mechanisms resulting from its mutation have not been elucidated. Given the same genetic location of the ZNF543 gene with TRIM28 and their effects on PD pathogenesis, we surmised that ZNF543 might act as a transcription factor for TRIM28 gene expression. By knocking out the ZNF543 gene via the CRISPR/Cas9 editing platform, we assessed the functional effect of loss of expression of this gene on TRIM28 gene expression. Four sgRNAs with different PAM sequences were designed against two parts of the regulatory region of ZNF543 gene, and highly efficient disruption of ZNF543 expression in human neuroblastoma cell line was evaluated by polymerase chain reaction and T7 endonuclease assay. Moreover, evaluation of TRIM28 gene expression in ZNF543-knocked-out cells indicated a significant increase in TRIM28 gene expression, suggesting that ZNF543 probably regulates the expression of TRIM28. This approach offers a window into pinpointing the mechanism by which ZNF543 gene mutations mediate PD pathogenicity.


Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disorder, with a prevalence of 0.3%, affecting approximately 1-2% of individuals over 60 years [1,2]. Recent studies have shown that familial PD genes act as starting points in the pathogenesis of PD. In addition to SNCA, which is the first Parkinson's disease-related gene, several risk loci associated with PD harbors common causative genes such as DJ-1, DNAJC6, EIF4G1, LRRK2, PARK2, PINK1, PLA2G6, and SYNJ1, have been identified for PD or other parkinsonism disorders [3,4]. In recent years, genome-wide association approaches such as exome and genome sequencing have investigated various PD candidate variants [5][6][7][8][9]. Using largescale whole-exome sequencing, Jansen et al. identified 27 riskassociated genes with loss-of-function variants in PD individuals. Genetic knockdown of the ZNF543 gene as a candidate disease-causing gene (by siRNA) showed a decrease in the mitochondria number per cell, indicating the role of this stop gain variant in PD pathology [6]. On the other hand, Rousseaux et al. demonstrated that α-synuclein accumulates within the nucleus through tripartite motif-containing 28 (TRIM28) protein [10,11]. Within the nucleus, α-synuclein binds to the mitochondrial transcription activator peroxisome proliferatoractivated receptor gamma-coactivator 1α (PGC1α) promoter, which leads to inhibition of PGC1α promoter and reduction of PGC1α mRNA and protein, resulting in mitochondrial dysfunction and reduced levels of mitochondrial biogenesis [12,13]. Inspired by the similar effects of knocking down the ZNF543 gene and overexpressing TRIM28 on PD and their genomic co-localization (chromosome 19q13.43), we reasoned 1 3 that they might be related to each other. ZNF543 is a member of the Kruppel-associated box (KRAB)-containing zinc finger proteins family, which function as transcription factors. Most of these factors are associated with the KRAB domain, an almost transcriptional inhibitor domain [14]. This fact and pathologically overlapping of ZNF543 and TRIM28 led us to ask whether ZNF543 acts as a transcription factor for TRIM28 gene expression.
Developing precise and scalable loss-of-function gene mutation technologies creates powerful tools for investigating the genes' function in protein signaling networks, disease mechanisms, and the developmental pathway activation [15,16]. In this regard, genetic screens can play an essential role in the unbiased discovery and phenotype characterization of specific genetic elements. Genome editing technologies based on the clustered, regularly interspaced, palindromic repeats (CRISPR) system as an RNA-guided platform, have generated a tremendous motivation in the manipulation of genomic information contents moving toward disease-gene association and individualized treatment decision making [17][18][19][20]. The type II CRISPR system from Streptococcus pyogenes consists of a duplex of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), which provides a potential platform for guiding Cas9 (CRISPR-associated endonuclease)-mediated cleavage. This system has been compatible with various eukaryote cell types and whole organisms to induce targeted mutations derived from DNA break repair of non-homologous end joining (NHEJ) or homologous directed repair (HDR). NHEJ can lead to insertions or deletions (indels) at the targeted site, resulting in a frameshift in the target sequence and thereby disrupting the gene [21,22].
Herein, hypothesizing that ZNF543 may act as a transcription factor for TRIM28 gene expression, by using the CRISPR/Cas9 editing platform, we assessed the functional effect of loss of the expression of ZNF543 on TRIM28 gene expression. Four sgRNA with different PAM sequences were designed against two parts of the regulatory region (5′-untranslated region (5'-UTR)) and the core promoter of the ZNF543 gene, and their knock-out efficiency for ZNF543 gene was evaluated. We achieved a highly efficient disruption of ZNF543 expression in the human neuroblastoma cell line. Moreover, evaluation of TRIM28 expression in ZNF543-knocked-out cells indicated higher levels of TRIM28 mRNA.

Bioinformatics Analysis
In order to design the synthetic sgRNAs, the sequence of the ZNF543 gene regulatory region, including a part of promoter and 5'-UTR, was obtained from the eukaryotic promoter database (https:// epd. epfl. ch) and the Ensembl database (https:// www. ensem bl. org). Four CRISPR RNAs (crRNAs) targeting the ZNF543 regulatory region named g1-g4 were designed according to the results of the online CRISPR design tool (http:// crispr. mit. edu) and were synthesized by Bioneer Co. (South Korea). The sense and antisense sequences of each crRNA were listed in Table S1.

Generation of sgRNAs and Cas9 Plasmids
The pSpCas9 (BB)-2A-puro (pX459) expression vector was a gift from Feng Zhang (Addgene plasmid # 48,139), which has a crRNA:tracrRNA (sgRNA), Cas9, and puromycin resistance genes. Synthetic sense and antisense strands were annealed to form the double-strand crRNA. crRNAs targeting the ZNF543 gene regulatory region were cloned into pX459 using the BbsI enzyme (Thermo Fisher Scientific (USA)), according to the previously published protocol [22]. The sgRNA cassette insertion into the expression vector was verified by colony PCR and Sanger sequencing by primer listed in Table S2, and the schematic map of primers used in colony PCRs was shown in Figure S1.

Cell Preparation and Transfection Experiments
The human neuroblastoma SH-SY5Y cells were provided by the Pasteur Institute of Iran. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, USA) with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin antibiotics (Gibco, USA) at 37 °C in a 5% CO2-air humidified atmosphere. The cells were separately seeded in six-well plates and were transfected with Px459 vectors containing the desired sgRNA and Cas9 sequences, using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY, USA). For inducing a deletion mutation, all plasmids (containing g1-g4) were co-transfected in another well of cells. After 48 h, puromycin (1 µg/ml) was added to the wells and cells were selected for the Cas9/ gRNA vector expression after another 48 h.

T7 Endonuclease Assay
In order to confirm the efficiency of the designed gRNAs for disruption of the regulatory region upstream of ZNF543 gene, T7 endonuclease cleavage assay was performed. To evaluate the mutation efficiency, each of the gRNAs (g1-g4) was transfected to a separate well of the plate, and total genomic DNA was isolated from 10 6 transfected cells using a TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). The regulatory region of the ZNF543 gene was first amplified using Phusion High-Fidelity DNA Polymerase (New England Biolabs) with primers amplifying an 828 bp fragment of the ZNF543 regulatory part containing the target sites of gRNAs (Table S2). PCR amplicons were purified using GeneJET PCR Purification Kit (Thermo Fisher Scientific). For T7 endonuclease assays, 400 ng of the purified PCR product was denatured and reannealed in 1X NEB buffer (New England Biolabs) in a total volume of 20 µl after which 10 U of T7 endonuclease (New England Biolabs) was added to the solution and incubated for 45 min at 37 °C and then the reaction stopped with 1 µl of 0.5 M EDTA. The products were then run on a 2.5% agarose gel. The mutation efficiency of gRNAs with different protospacer adjacent motifs (PAMs) was evaluated by quantifying DNA signals using ImageJ software (http:// rsb. info. nih. gov/ ij/).

Detection of Deletion Mutation in ZNF543 and DNA Sequencing
To generate deletion mutations in the regulatory region of ZNF543 gene, all plasmids (pX459-g1-g4) were co-transfected into the SH-SY5Y cells. As aforementioned, total DNA was extracted, and target regions were amplified using Phusion High-Fidelity DNA Polymerase and primers listed in Table S2. The amplification products were run on a 1.5% agarose gel and then purified using a column-based precipitation kit (Bioneer). The purified PCR fragments related to control and transfected cells were submitted for Sanger sequencing to investigate the deletion mutation (Bioneer, South Korea).

Relative Quantification of ZNF543 and TRIM28 Gene Expression
To evaluate the efficiency of designed gRNAs (g1-g4) for creating the deletion mutation in ZNF543, reverse transcriptase (RT)-PCR was performed. Total RNA was extracted from control and transfected cells using Trizol reagent (Zaver Zist Azma, Iran). cDNA was synthesized from 2 μg of the total RNA by SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). The cDNA products equivalent to 200 ng of total RNA were used as the template in a 25 μl PCR system. Moreover, the relative mRNA expression of ZNF543 and TRIM28 genes was evaluated by quantitative real-time PCR (qRT-PCR) using SYBR Green P n CR Master Mix (SMObio, Taiwan). Expression levels of genes of interest in cells transfected by different gRNAs were normalized against GAPDH mRNA as an endogenous control. Primer sequences used in these assays were listed in Table S2.

Rescue Assay
For performing a rescue assay, the coding region of ZNF543 cDNA was amplified by PCR from control cells cDNA using ZaF and ZaR primer (Table S2), which includes EcoR1 and BamH1 enzyme site, respectively, the PCR product and pCDNA3-GFP expression vector(addgene plasmid #74,165) be co-digested with EcoR1 and BamH1 enzyme. The ligation process was done the following. 80 ng purified digest PCR product and 30 ng linear plasmid were added to 1X ligase buffer in a total volume of 20 µL. The ligation process was started by adding 10 U of ligase enzyme and incubating at 22 °C for 2 h.
Rescue experimental cultures were transfected with 800 ng of recombinant pCDNA3-GFP plasmid at a density of 10 6 cells(ZNF543 -/-)/ml by lipofectamine 2000 reagent. Control cells were treated with the same concentration of empty plasmid, and after 24 and 48 h, cells were harvested for RNA extraction and cDNA synthesis. The relative mRNA expression of ZNF543 and TRIM28 genes was evaluated by quantitative real-time PCR, and different expression of genes was normalized against GAPDH mRNA as an endogenous control.

Statistical Analysis
The data are presented as mean ± SD from at least three independent experiments. P-values of < 0.05 were considered as the criterion for the statistical significance. Gene expression data were conducted by one-way ANOVA followed by Tukey's post hoc test using the SPSS software (version 24).

Generation of gRNAs Expressing Plasmids
To selectively disrupt the ZNF543 gene expression in human neuroblastoma SH-SY5Y cells, four sgRNAs (g1-g4) targeting a part of the promoter and the 5'-UTR of ZNF543 gene (Position: chr19:57,831,623-57,832,069 = ∆447 bp), were designed and selected based on the prediction of high on-target and low off-target effects (Fig. 1a). Synthetic sense and antisense strands were annealed to form a double-stranded DNA with sticky ends (Fig. 1b) and ligated into pSpCas9 (BB)-2A-puro (pX459) expression vector linearized with BbsI restriction enzyme. Colony PCR and Sanger sequencing results using CRISPR-Cas9 specific primers confirmed the correct insertion of g1-g4 into the pX459 expression vectors (Figs. 1c and 1d).

Detection of the Deletion Mutation in the Regulatory Region of ZNF543 Gene
Gene editing efficiency of the pSpCas9 (BB)-2A-puro-g1-g4 (pX459-g1-g4) system was evaluated in the SH-SY5Y cells.
To generate deletion mutations in the regulatory region of ZNF543 gene, all plasmids (pX459-g1-g4) were co-transfected into SH-SY5Y cells. Un-transfected cells were used as the control. After 48 h, puromycin (1 µg/ml) was added to the cells, and cells were assayed for the Cas9/gRNA vector expression after another 48 h. In order to detection of the deletion mutations, the genomic DNA of un-transfected cells, puromycin-untreated transfected cells, and puromycintreated transfected cells were used as the template for PCR reactions by primers listed in Table S2. In Fig. 2a, Lane 1, un-transfected cells showed an 828 bp band on the agarose gel corresponding to the wild-type ZNF543 gene target site. Puromycin-untreated transfected cells represented both bands of 828 bp and 381 bp corresponding to the wild-type and mutated ZNF543 gene target site, respectively (Fig. 2a,  lane 2). Transfected cells treated with puromycin exhibited a characteristic 381 bp band on the agarose gel, representing the deletion mutation of the regulatory region of the ZNF543 gene in these cells (Fig. 2a, lane 3). These results imply that the pX459-g1-g4 system has high efficiency for generating deletion mutations in a part of the regulatory region of the ZNF543 gene in SH-SY5Y cells. The sequencing results of PCR products were also entirely consistent with our prediction for the deletion mutations in the ZNF543 gene target site. As shown in Fig. 2b, the deletion mutation extends from −235 to + 212 of the ZNF543 gene, including a GC-box and a CCAAT-box positioned in the −30 and −79 regions of the target site, respectively.

T7 Endonuclease Assay
Using the T7 endonuclease cleavage assay, we analyzed targeted mutagenesis in transfected cells. DNA extracted from un-transfected and transfected cells with pX459-g1-g4  Table S2, 1-3) pX459-g1, 4-6) pX459-g2, 7-9) pX459-g3, 10-12) pX459-g4, M: DNA marker. d Sequencing results of pX459-g1-g4 expression vectors with specific vector primers listed in Table S2 plasmids were subjected to PCR and T7 endonuclease cleavage. T7 endonuclease assay using the PCR products was performed. The assay showed cleavage bands of the ZNF543 regulatory region in transfected cells with pX459-g3 and pX459-g4 plasmids (Fig. 3a). The assay in transfected cells with pX459-g1 and pX459-g2 plasmids showed that cleavage bands are very close to the control bands. This can be due to the position of the g1 and g2 target sites at the beginning of the PCR products. The mutation percentages of each gRNA construct with the different PAM sequences were estimated from band intensities using Image J (Fig. 3b). Investigating signals of DNAs by ImageJ showed GGG and CGG PAM sequences next to the targeted sites by g1 and g3, respectively, are more efficient in the mutation generation (P-value < 0.01) compared to TGG and GGG PAM sequences next to the targeted sites by g2 and g4 (P-value < 0.05), respectively. These results highlight that the pX459-g1-g4 system can generate the indel mutation in an efficient manner in SH-SY5Y cells.

Relative Quantification of ZNF543 and TRIM28 Gene Expression
To evaluate the efficiency of designed gRNAs (g1-g4) for ZNF543 gene disruption in SH-SY5Y cells, reverse transcriptase-PCR (RT-PCR) was performed. As displayed in Fig. 4a, RT-PCR products of total RNA extracted from cotransfected cells with pX459-g1-g4 plasmids show no band on the agarose gel compared to un-transfected cells. Moreover, the relative mRNA expression of the ZNF543 gene was evaluated by quantitative real-time PCR (qRT-PCR). As shown in Fig. 4b, the expression of gRNAs targeting the regulatory region of the ZNF543 gene in SH-SY5Y cells significantly decreased the ZNF543 gene expression (P-value < 0.05). g1 and g2 had a slight effect on decreasing the ZNF543 expression (P-value < 0.05), while g3 and g4 decreased it by more than 75% (P-value < 0.01). The mRNA levels of ZNF543 decreased by more than 95% when the regulatory region was targeted with four gRNAs, indicating that a mix of all pX459-g1-g4 plasmids has higher efficiency for ZNF543 gene disruption. Hence, for evaluating the effect of ZNF543 gene disruption on TRIM28 gene expression, g3 and a mix of all four gRNAs were used for the following experiments. Co-transfected cells with pX459-g3 and the combination of all pX459-g1-g4 plasmids represented a significant expression change for TRIM28 expression compared to untransfected cells (P-value < 0.05), which indicates a possible role of ZNF543 for TRIM28 gene expression (Fig. 4c).

Rescue Assay
The rescue assay was performed to ensure the effectiveness of the ZNF543 protein on the expression of the TRIM28 gene. As shown in Fig. 5c, after normalization of the gene expression data to the endogenous control, the relative mRNA expression of the ZNF543 gene was increased    g1,g2,g3,g4,mix-g1-g4 suppress ZNF543 gene about 35% P = .048, 46% P = 0.041, 83% P = 0.006, 71% P = 0.007 and 98% P = 0.004, respectively. c TRIM28 gene expression in transfected cells with pX459-g3 and a mix of all pX459-g1-g4 plasmids and un-transfected cells (control) (overexpress of TRIM28 during treatment by g3 and mix g1-g4 was 2.9 fold P = 0.008, 3.1 fold P = 0.006, respectively). Each point represents the mean value ± SEM for at least three independent observations (*P < 0.05, **P < 0.01 versus control group) eightfold in p-CDNA3-GFP-ZNF543 recombinant vector treated cells (P-value = 0.006). About 40% suppression TRIM28 gene resulted from overexpression of ZNF543 (P-value = 0.043), which shows the connection between ZNF543 protein and the expression of the TRIM28 gene. The result was confirmed in three repeat wells, and the experiment was carried out 3 times.

Discussion
Recently, genome-wide association studies emphasized the association of loss-of-function (LoF) variants with Parkinson's disease (PD). By integrating whole-exome sequencing data and available evidence, Jansen et al. have suggested that an LoF variant of the ZNF543 gene is a candidate for PD. They showed that knocking down of ZNF543 gene can decrease the mitochondria number per cell, indicating the role of this variant in PD pathology [5,6]. ZNF543 is a zinc finger protein containing the KRAB domain, a transcriptional inhibitory domain [14]. There is no evidence for the ZNF543 function in PD, and mechanisms resulting from its mutation were not elucidated. Given the role of TRIM28 in mitochondrial dysfunction and reduced levels of mitochondrial biogenesis in PD, as well as its same location as the ZNF543 gene on chromosome 19 (19q13.43), we surmised that ZNF543 might act as a transcription factor for TRIM28 gene expression [23]. Also, previous studies have shown that this chromosomal location (19q13) is associated with nervous system disorders [24]. To test whether ZNF543 itself regulates the levels of TRIM28 gene expression, we knocked out the ZNF543 in the human neuroblastoma SH-SY5Y cells via CRISPR/Cas9 technology. To disrupt ZNF543 gene expression in SH-SY5Y cells, we designed four gRNAs that target a part of the promoter and the 5'-UTR of the ZNF543 gene. The deletion mutations in the regulatory region of ZNF543 gene were verified by Sanger sequencing, PCR, and T7 endonuclease assay. To evaluate the efficiency of each designed gRNA (g1-g4) for ZNF543 gene disruption in SH-SY5Y cells, RT-PCR and qRT-PCR were performed. The results showed that the best efficiency for ZNF543 gene disruption was obtained by g3 and g4, which indicates that the 5'-UTR of the ZNF543 gene is a critical regulation point for its expression. We proved that a mix of all g1-g4 can create sufficiently high levels of ZNF543 gene disruption, yielding the output of NHEJ. To address the impact of ZNF543 disruption on TRIM28 gene expression, we performed qRT-PCR for TRIM28 mRNA in the ZNF543knocked out SH-SY5Y cells. We found that the disruption of the ZNF543 gene can significantly increase TRIM28 gene expression (P = 0.006), suggesting that ZNF543 probably regulates the expression of the TRIM28 gene. The rescue experiment reinforced this hypothesis, and the expression of ZNF543 cDNA using pcDNA3-GFP plasmid in the cell line model showed a significant decrease in TRIM28 mRNA expression (P = 0.043). These results offer a window into understanding the pathogenicity of the stop gain variant of the ZNF543 gene in PD via the regulation of TRIM28 gene expression, which is in keeping with previous studies [10,23]. α-synuclein accumulates as a significant component of the Lewy body, plays an essential role in Parkinson's disease and other neurodegenerative diseases. α-synuclein aggregation and disease progression can result from gene multiplications and gene overexpression or nucleotide polymorphisms in the SNCA gene. Some recent studies have targeted the SNCA gene as one of the more critical genes in PD therapy [25,26]. The previous research demonstrates α-synuclein accumulation results from overexpression of TRIM28 protein [10,23]. Therefore, suppressing TRIM28 protein at the mRNA level is a more reasonable target in PD therapy. Due to the essential role of TRIM28 protein in the cell process [27,28], and the temporal nature of some methods for protein knocking down, it isn't suggested TRIM28 gene be directly targeted. Given that this study shows downregulated TRIM28 gene by ZNF543 gene expression, overexpression of the ZNF543 gene can be an effective method in PD therapy (Fig. 5c). However, elucidating specific mechanisms resulting from ZNF543 mutations and their relation with TRIM28 and a-Syn require clarification in future studies.