Targeted RP9 ablation and mutagenesis in mouse photoreceptor cells by CRISPR-Cas9

Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells. Genetic mutations in many spliceosome genes confer human eye diseases. Mutations in the pre-mRNA splicing factor, RP9 (also known as PAP1), predispose autosomal dominant retinitis pigmentosa (adRP) with an early onset and severe vision loss. However, underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown. Here, we utilize the CRISPR/Cas9 system to generate both the Rp9 gene knockout (KO) and point mutation knock in (KI) (Rp9, c.A386T, P.H129L) which is analogous to the reported one in the retinitis pigmentosa patients (RP9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells in vitro. We found that proliferation and migration were significantly decreased in the mutated cells. Gene expression profiling by RNA-Seq demonstrated that RP associated genes, Fscn2 and Bbs2, were down-regulated in the mutated cells. Furthermore, pre-mRNA splicing of the Fscn2 gene was markedly affected. Our findings reveal a functional relationship between the ubiquitously expressing RP9 and the disease-specific gene, thereafter provide a new insight of disease mechanism in RP9-related retinitis pigmentosa.

Herein, we successfully generated Rp9 gene knockout (Rp9-KO) and point mutation knock in (Rp9-KI) (Rp9, c.A386T, P.H129L) which was analogous to the reported one in the retinitis pigmentosa patients (Rp9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells by using CRISPR/Cas9-mediated approach. Based on this cell model, we observed significant change in cell property and down regulation of RP associated genes, Fscn2 and Bbs2. Furthermore, we elucidated that pre-mRNA splicing of the Fscn2 gene was remarkably reduced in the mutated cells. Our study for the first time revealed a functional relationship between Rp9, the general splicing factor, and FSCN2, the photoreceptor-specific gene, and provided a new insight of disease mechanism in Rp9-causing retinitis pigmentosa.

Validation of cone photoreceptor-specific markers in 661 W Cells.
To test 661 W cell line whether is rod photoreceptor or cone photoreceptor, cells were immunostaining for rhodopsin, blue opsin, red or green opsin and cone arrestin. The results showed that 661 W cells were positive with the cone photoreceptor-specific markers, red or green opsin (Fig. 1A), blue opsin (Fig. 1B) and cone arrestin (Fig. 1C), but negative with rod photoreceptor-specific marker, rhodopsin (Fig. 1D). These results verified that 661 W cells were cone photoreceptor originated.
Site-specific DNA cleavage executed by Cas9/sgRNA in 661 W cells. To test whether Cas9/sgRNA could cleave targeted regions of Rp9, we designed two sgRNAs targeting different regions of Rp9 gene ( Fig. 2A). Each Cas9/sgRNA was transfected into 661 W cells and a T7EI assay was performed to determine the cutting Figure 1. Immunocytochemical staining of 661 W cells. Cells grew on glass cover slips and were fixed with cold 4% PFA, immunolabeled with primary antibodies against red/green opsin (A), blue opsin (B), cone arrestin (C) and rhodopsin (D). DAPI (blue) was used to detect the nuclei. Scale bar: 25 μ m. efficiency of each sgRNA. The results indicated that sgRNA-1 and sgRNA-2 designed to target the Rp9 gene were highly active, inducing mutations at frequencies of 23% for sgRNA-1 and 19% for sgRNA-2 (Fig. 2B). Furthermore, no mutation was detected using the T7EI assay when cells were transfected with Cas9 plasmid alone. Taken together, these results suggested that sgRNA-1 and sgRNA-2 efficiently targeted the Rp9 and led to NHEJ-mediated indels at target sites.
Generation of Rp9-KI and Rp9-KO in 661 W cells. Encouraged by the high efficiency of Cas9/sgRNAs, we explored whether these Cas9n pairs could catalyze site-specific DNA cleavage and HDR in 661 W cells. 661 W cells were transfected with donor vector and Cas9n pairs targeting the exon 5 of Rp9 gene. 3days later, transfected cells were selected for further 7 days with 1.0 mg/ml G418. After selection, cells were digested and 500 cells were transferred to a 10-cm dish. 2 weeks later, clones were picked out and propagated for another week. After propagation, 22 clones were randomly selected for genotyping. The genomic DNA of 22 clones was analyzed by PCR amplification with specific primers showed as Supplemental Table 1. Figure 2C illustrated that clone 5 contained two different amplification products whereas other clones contained only one amplification product. Sequencing result of the upper bands of clone 5 revealed the point mutation, c.A386T (Fig. 2D). The result demonstrated that the clone 5 was a heterozygous Rp9-KI cell. Also, we sequenced the other 21 clones, the results showed that five clones were heterozygous Rp9-KO cells which only one allele was mutated with nucleotide indels thus causing a frameshift mutation. Thus Clone 12 would represent for Rp9-KO (Fig. 2D).

Decreased proliferation and migration by Rp9-KI and Rp9-KO. We sought to detect whether these
Rp9 mutations had any biological effects on 661 W cells. Rp9-KI and Rp9-KO showed inhibition of cell growth as compared with control based on the MTT assay (Fig. 3A). A reduction in growth rate was detected at day 3. At day 4, the growth rate decreased 27.6% in Rp9-KI and 25.6% in Rp9-KO cells compared with control group (n = 3, P < 0.001, Fig. 3A). Western blot analysis confirmed that proliferation-related gene Ccnd2 expression was remarkably reduced in Rp9-KI and Rp9-KO cells (Fig. 3B). We then performed in vitro scratch assay to examine whether Rp9 mutations were involved in the regulation migration of 661 W cells. A dramatic migration reduction was observed in Rp9-KI and Rp9-KO cells (Fig. 3C).

RNA-seq and differential expression analysis of Rp9-KI and Rp9-KO cells. Three libraries
were generated from 661 W, Rp9-KI and Rp9-KO groups and summaries of RNA-Seq analyses are shown in Supplement Table 3. About 42.66 (661 W), 38.56 (Rp9-KI), and 39.83 (Rp9-KO) million clean reads were obtained for each transcriptome. The Q30 scores (the average quality value) were above 93%. The RNA-Seq raw reads have been submitted to the NCBI SRA database (accession number: SRR5131155, SRR5131263, and SRR5131264).
Through RNA-Seq, a total of 14006, 13885 and 13226 expressed genes were detected in 661 W, Rp9-KI and Rp9-KO cells respectively. Among them, 784 and 934 genes were differentially expressed in Rp9-KI and Rp9-KO cells compared with 661 W cells (Fig. 4A,B). Overlap of differentially expressed genes containing down-regulated RP associated gene, Fscn2, identified by Rp9-KI and Rp9-KO cells were presented (Fig. 4C). We used qRT-PCR to confirm the expression of Fscn2 and found it to be markedly down-regulated in Rp9-KI and Rp9-KO cells (Fig. 5A). We also used qRT-PCR to quantify other RP associated genes which were significant down-regulated RP9 mutagenesis affects pre-mRNA splicing of photoreceptor gene FSCN2. Previous report showed that PRPF31 mutation inhibited splicing of retina-specific gene, RHO 25 . In order to confirm whether RP9 mutation inhibited retina-specific splicing substrate genes expression, we selected Fscn2 and Bbs2 for the further experiments. Fscn2 and Bbs2 pre-mRNA splicing were examined using RT-PCR with specific primers (Fig. 5B, Supplemental Table 1). As shown in the Fig. 5C, Fscn2 splicing was significantly inhibited in Rp9-KI and Rp9-KO compared with control group. Quantification of splicing efficiency obtained from three independent experiments was shown in Fig. 5D. However, Rp9-KI and Rp9-KO did not affect the Bbs2 splicing, because there was no significant change in the ratio of pre-mRNA in splicing products (Fig. 5D).

Discussion
We have demonstrated that RP9 patient-specific rod photoreceptors conferred degeneration in vitro by using patient-specific induced pluripotent stem cells 26 . However, underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains unknown. Pre-mRNA splicing is crucial for the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited genes 27 . Mutations that interfere with splicing play an important role in human eye disease [28][29][30] . Here we have shown that proliferation and migration significantly decrease in the Rp9 mutant 661 W photoreceptor cells. RP associated genes, Fscn2 and Bbs2, both are markedly down-regulated in the mutated cells. Further investigation indicated that pre-mRNA splicing of the Fscn2 gene was markedly reduced. In this study, we successfully generated Rp9-KI (HDR) with an efficiency of 4.5% and Rp9-KO (Indels) with an efficiency of 22.7%, which was consistent with reports from other groups. For example, Mali et al. reported 10-25% indel rates in 293 T cells in 2013 20 . Likewise, in mouse embryonic stem cells study, the efficiency of NHEJ-mediated knock-out was 28-50%, whereas the efficiency of HDR-mediated knock-in was below 10% 23 . Importantly, Rp9-KI and RP9-KO resulted in inhibition of cell proliferation and migration.
Previous work by Yuan et al. demonstrated that PRPF31 mutation could inhibit RHO splicing 25 . Through RNA sequencing, we confirmed the expression of Fscn2 markedly down-regulated in Rp9-KI and Rp9-KO cells. Fscn2, actin-bundling proteins, is a photoreceptor-specific protein of fascin family, which plays an important role in photoreceptor disk morphogenesis. A frame-shift mutation in FSCN2, 208delG, was reported in Japanese adRP patients 31 . A mouse model carrying a targeted disruption of Fscn2 showed progressive photoreceptor degeneration 32 .
RT-PCR results showed that the 661 W cell had normal pre-mRNA splicing of Fscn2, whereas Rp9-KI and Rp9-KO cells inhibited this splicing progress. Our results were consistent with those recently reported by Mordes et al., who found dominant-negative effect of PRPF31 mutation on FSCN2 pre-mRNA splicing 33 . However, Maita et al. reported that RP9 p.H137L mutation had no effect on E1A splicing 34 . This might suggest that Rp9 only inhibits a fraction of gene splicing. Our experiments revealed a functional link between ubiquitously expressed Rp9 gene and the expression of the retina-specific gene, Fscn2. Our results that Rp9-KI and Rp9-KO blocking pre-mRNA splicing of Fscn2 provided a new insight for the photoreceptor-specific phenotype of RP9 mutations. Defected Fscn2 gene products may contribute to photoreceptor cell death.
We also detected the RP associated gene, Bbs2, markedly down-regulated in Rp9-KI and Rp9-KO cells. Our result showed that Rp9-KI and Rp9-KO did not affect the Bbs2 splicing. It demonstrated that not all splicing events were equally sensitive to Rp9 mutation. Rp9 mutants may inhibit pre-mRNA splicing of a subset of photoreceptor genes, such as Fscn2 intron 3 and intron 4, but not other splicing events such as Bbs2 intron 8 and intron 10. In summary, we have shown that Rp9 mutations obviously inhibit cell proliferation and migration. Furthermore, the splicing of adRP associated gene, Fscn2, is also significantly inhibited in Rp9 mutation cells. Our results on RP9 provide an explanation why the mutations in the ubiquitously expressed splicing factors can cause adRP.
Cell culture. Cone-derived cell line (661 W) originated from a transgenic mouse line with retinal tumor 35 was cultured in DMEM medium (Gibco, Carlsbad, USA) supplemented with 10% heat-inactivated FBS (Gibco, Carlsbad, USA)and 100 μ g/mL penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO 2 . 661 W cells were plated into 6-well plates for transfection. After twenty four hours, cells were replaced with new complete medium and the DNA mixed with FuGENE HD Reagent (Roche, Basel, Switzerlands) in Opti-MEM (Gibco, Carlsbad, USA) according to the manufacturer's manual. For G418 selection, 661 W cells transfected with px335-mRp9sg1, px335-mRp9sg2 (Cas9n pairs) and donor vector were selected with 1 mg/ml of G418.
Genomic DNA isolation, amplication and T7EI assay. To validate the mRp9 sgRNAs, the genomic DNA of each Cas9/gRNA-transfected cells was extracted using the Blood/Cultured cells DNA Kit (Simgen Biotech Co.Ltd, Hangzhou, China) following the manufacturer's instruction. The regions containing thetarget sites were amplified by PCR using Phanta Max Super-Fidelity DNA Polymerase with gene-specific primers (Supplemental Table 1) under the following conditions: 95 °C for 3 min; 30 cycles (95 °C for 15 s, 58 °C for 15 s, 72 °C for 30 s) and 72 °C for 5 min.
The T7EI assay was performed according to the manufacturer's instructions. In brief, 20 μ l of PCR product was denatured and annealed by heating at 95 °C for 5 min and ramped down to 25 °C at 6 °C/min. Then, 5 μ l annealed samples, 1.1 μ l NEBuffer 2 (10x), 0.5 μ l T7EI and 4.4 μ l ddH 2 O were added together and incubated at 37 °C for 30 min. Cleaved DNA fragments were separated on 1.5% agarose gels and the DNA concentration of each band was quantified using the ImageJ software. Percent values of indels were calculated as described 15 . For genotyping PCR, genomic DNA was amplified using 2 × Super Taq PCR MasterMix (BioTeke Corporation, Beijing, China) with primers listed (Supplemental Table 1).
Immunocytochemistry. 661 W cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes. Cells were permeabilized and blocked in PBS containing 4% BSA and 0.5% Triton X-100 for 1 hour at room temperature, incubated overnight with primary antibody at 4 °C, and then subjected to immunohistochemistry as previously described 36 .
Cell proliferation assay. A total of 5 × 10 3 661 W cells were seeded in 96-well plates with 100 μ l DMEM containing 5% FBS and 100 μ g/mL penicillin/streptomycin. Cell proliferation was assessed by MTT assay. MTT assay was carried out according to the method by Mosmann 37 . Briefly, 10 μ l of MTT solution was added into each well and the cells were incubated for 4 hours. Then medium was discarded and supplied with 150 μ l DMSO. Finally, Cell proliferation was assessed by measuring the absorbance at 490 nm using Spectra Max M5 (Molecular Devices, Sunnyvale, CA, USA).
In Vitro scratch assay. In Vitro Scratch Assay was performed as previously reported 38 . Simply, cells were scratched with a 200 μ l pipet tip. Remove the debris by washing the cells once with 1 ml of DPBS and then replace with 2 ml DMEM supplemented with 2% FBS and 100 μ g/mL penicillin/streptomycin. Photograph was taken immediately after scratching and at 24 or 48 hours after culture. The ability of migration was evaluated by comparing with the migration rate in the center of the gap.
Western blot analysis. Total protein extracts were prepared using cell lysis buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 mM leupeptin, 1 mM pepstatin, 80 mM aprotinin). Protein contents were quantified by the Bradford reagent according to the manufacturer's instructions. Equal amounts of proteins were separated by 12% SDS-PAGE and transferred to a nitrocellulose blotting membrane (PALL Corporation, Port Washington, USA). Membranes were blocked for 1 h in 1 × TBS containing 10% non-fat milk, 0.1% Tween 20 and incubated overnight with primary antibodies: rabbit anti-Ccnd2 (Santa Cruz Biotechnology, Santa Cruz, USA), mouse anti-Gapdh (KangChen Bio-tech Inc., Shanghai, China). Then membranes were incubated with IRDye ® 680-conjugated goat anti-rabbit, IRDye 800CW-conjugated goat anti-mouse. Fluorophore-conjugated antibodies were detected using the Odyssey ® Imager (LI-COR Biosciences Inc., Lincoln, USA).
RNA library construction and sequencing. RNA  Isolation Module. The isolated mRNA was fragmented and used to synthesize the first cDNA. Second strand cDNA synthesis was generated using DNA Polymerase I and RNase H. The double-stranded cDNAs were purified by Agencourt AMPure XP system (Beckman Coulter, Brea, USA) and subjected to end repair and adapter ligation. The ligation products were enriched by PCR amplification and purified using Agencourt AMPure XP system. Sequencing reactions were carried out on the Illumina HiSeq 2500.
Transcriptome analysis and identification of differential gene expression. The raw reads were firstly processed through in-house perl scripts. Clean reads were obtained by removing reads containing adapter sequences, unknown nucleotides> 5%, low quality reads. The clean reads were mapped to mouse genome (mm10) with TopHat2 39 . Gene expression levels were estimated using fragments per kilobase of exon per million fragments mapped (FPKM).
Prior to differential gene expression analysis, the read counts of each sequenced library were adjusted by edgeR program package 40 . DEGseq 41 was performed to evaluate differential expression between control, Rp9-KI and Rp9-KO groups. The false discovery rate (FDR) control method was applied to define the threshold of the P-value to compute the level of significance. Significantly differential expression was accepted as |log2FC| > 1 and FDR < 0.05.

RT-PCR.
For reverse transcription, isolated RNA was treated with DNase I (Thermo Scientific, Rockford, USA) according to the manufacturer's manual. The DNase I-treated RNA was transcribed into cDNA using MMLV Reverse Transcriptase (Promega, Madison, USA). The cDNA was subsequently used as template to perform RT-PCR and qRT-PCR.
qRT-PCR was performed as described in the method of Fast start universal SYBR Green Master (Roche Molecular Biochemicals, Mannheim, Germany) with 7500 Real-Time PCR System. Gapdh expression was used for normalization. The specificity of PCR products was verified by melt curve analysis. Sequences of primers are shown (Supplemental Table 1).
Statistical analysis. Data were expressed as means ± SEM and analyzed by two-way analysis of variance (ANOVA) with GraphPad. Differences were considered to be statistically significant at a P value of 0.05 or less.