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. 2025 Dec 18;21(1):102747. doi: 10.1016/j.stemcr.2025.102747

Human CRX regulates photoreceptor cells development via bidirectional transcriptional control in retinal organoids

Yuan Wang 1,4, Bingbing Xie 1,2,4, Xiaojing Song 1,4, Guanjie Gao 1, Yuanyuan Guan 1,3, Dandan Zheng 1, Ping Xu 1, Xiufeng Zhong 1,5,∗
PMCID: PMC12925966  PMID: 41418782

Summary

CRX (cone-rod homeobox) is a key regulator of retinal photoreceptor development, yet its human-specific functions remain poorly understood due to scarce human retinal tissues and significant species differences. Here, we established a human CRX-mCherry fluorescent reporter retinal organoid (RO) model to dissect CRX-mediated gene regulation. Using FACS, RNA sequencing, and Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, we identified CRX target genes and revealed its dual regulatory role: it activates photoreceptor-specific genes (e.g., RP1L1, linked to inherited retinal degeneration) in a dose-dependent manner, while suppressing non-photoreceptor genes (e.g., PCDH8 and PROX1). Notably, we first generated the human CRX CUT&Tag dataset, providing direct insights into CRX’s genome-wide regulatory landscape in photoreceptor cell development. These findings demonstrate that CRX functions as both a transcriptional activator and repressor, ensuring photoreceptor-specific gene expression and preventing aberrant cell fate transitions. Our study provides critical insights into the role of human CRX in retinal development and implications for retinal degenerative diseases.

Keywords: CRX, retinal organoids, photoreceptor development, transcriptional regulation, cell fate determination

Graphical abstract

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Highlights

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    Dual regulatory role of human CRX in photoreceptor cell development

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    Generation of the first human CRX-bound CUT&Tag-seq dataset from human retinal tissue

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    Both CRX-mediated chromatin-binding and gene expression profiles of photoreceptor cells

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    Establishment of a human CRX-mCherry reporter retinal organoid model

Zhong and colleagues generated the first human CRX-bound CUT&Tag dataset and revealed that CRX governs photoreceptor development via bidirectional transcription: It activates photoreceptor-specific genes (e.g., RP1L1) and represses non-photoreceptor genes (e.g., PCDH8, PROX1). CRX knockout causes aberrant expression of PROX1(a horizontal cell-specific transcription factor), with distinct target specificities between human CRX and mouse Crx.

Introduction

In the retinal photoreceptor cells of mammals, a set of highly expressed transcription factors (TFs) is integral to their development and functional maintenance, with particular emphasis on specific TFs. For instance, RXRγ and THRB are predominantly expressed in cone photoreceptors (Deeb, 2006; Ng et al., 2001), while NRL and NR2E3 are primarily associated with rod photoreceptors (Mears et al., 2001; Milam et al., 2002). Additionally, OTX2 and CRX are expressed across all photoreceptor types (Chen et al., 1997; Furukawa et al., 1997; Nishida et al., 2003). Dysfunction of these TFs can lead to retinal diseases (Corbo and Cepko, 2005; Daniele et al., 2005; Ng et al., 2001; Roberts et al., 2005; Sato et al., 2007; Tran et al., 2014; Yamamoto et al., 2020), as they regulate a multitude of target genes, many of which are linked to retinal degenerative diseases (Hsiau et al., 2007). This establishes a gene expression network that is unique to photoreceptor cells (Hennig et al., 2008). A comprehensive understanding of these regulatory networks is essential for elucidating the mechanisms underlying retinal diseases.

CRX is one of the most important TFs during retinal development (Hennig et al., 2008; Sun and Chen, 2023; Swaroop et al., 2010). The expression of CRX occurs after that of OTX2 (Nishida et al., 2003), playing a crucial regulatory role in the development of photoreceptor cells and in maintaining their maturing state (Furukawa et al., 1997; Pan et al., 2023; Ruzycki et al., 2018). CRX regulates the development of retinal photoreceptor cells and is a common causative gene for inherited retinal diseases (IRDs) (Freund et al., 1997). Variations in CRX can lead to IRDs with different clinical phenotypes, including Leber congenital amaurosis (LCA), cone-rod dystrophy (CoRD), and retinitis pigmentosa (RP) (Tran et al., 2014). These diverse clinical phenotypes may be attributed to the characteristics of the CRX protein structure. CRX contains two important functional domains: the DNA binding domain (DBD) and the transactivation domain (TAD) (Freund et al., 1997; Tran et al., 2014). The DBD comprises a highly conserved homeodomain (HD) that demonstrates the capability to bind to DNA, whereas the TAD domain can recruit other coactivators (Corbo et al., 2010; Freund et al., 1997; Hennig et al., 2013; Peng et al., 2005; Peng and Chen, 2007; Tran et al., 2014). These two domains function synergistically to facilitate transcriptional activation. Over the past 2 decades, research has encompassed comprehensive investigations into the regulatory functions of CRX. This includes an in-depth analysis of CRX-bound elements (CBEs) (Chen et al., 1997; Corbo et al., 2010; Friedman et al., 2021; Ruzycki et al., 2018) as well as the regulatory impacts of CRX on its target genes. Recent studies have demonstrated that CRX functions not only as a transcriptional activator but also as a repressor (Hsiau et al., 2007; Peng et al., 2005; Pittler et al., 2004; White et al., 2016). By repressing cone-specific gene expression and activating rod-specific gene expression, CRX plays a crucial role in regulating the differentiation fate of rod photoreceptor cells (Hsiau et al., 2007; Peng et al., 2005; Pittler et al., 2004).

Previous studies have been largely constrained to animal models due to the scarcity of human retinal target tissues. Notably, although CRX is highly conserved in mammals, its spatiotemporal expression patterns differ between animals and humans (Bibb et al., 2001). There are several amino acid differences in DBD or TAD of CRX protein between these animal models and humans (Chen et al., 1997), which may result in distinct regulatory mechanisms for target genes, according to the latest studies (Shepherdson et al., 2024a, 2024b; Zheng et al., 2023, 2025). Consequently, it is imperative to conduct further research into the functional roles of human CRX.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for studying protein-DNA interactions and has been widely applied to research TF interactions (Johnson et al., 2007), including those involving CRX. However, ChIP-seq typically requires a substantial cell input, exhibits a high background signal, resulting in a low signal-to-noise ratio and reduced resolution, which makes it susceptible to false positives. In contrast, the recently developed CUT&Tag (Cleavage Under Targets and Tagmentation) technology offers several advantages, such as requiring fewer cells and providing a high signal-to-noise ratio, making it particularly suitable for studies on a limited input (Kaya-Okur et al., 2019). Over the past decade, retinal organoids (ROs) derived from human iPSCs (hiPSCs) have been extensively utilized to investigate retinal diseases (Chirco et al., 2021; Kruczek et al., 2021; Xu et al., 2023). In this study, we utilized CUT&Tag sequencing (CUT&Tag-seq) and employed human RO tissues to examine the regulatory function of human CRX in photoreceptor development, thereby elucidating its role in comparison to animal models.

Results

Generation and characterization of CRX-mCherry reporter hiPSC line

To prevent the activity of the CRX protein from being affected by the inserted reporter gene, we utilized a Porcine teschovirus 2A self-cleaving peptide (P2A) inserted between CRX Exon 4 and mCherry to create a non-fusion expression cassette (Figure S1A). We employed a donor plasmid containing a left homology arm (LA) of 551 base pairs (bp) and a right homology arm (RA) of 545 bp for targeting (Figure S2). A primer pair, CRX-VF and CRX-VR, was designed to target the region upstream of LA and RA, respectively (Figure S1A). A single round of PCR was conducted to confirm fragment insertion and homozygosity. PCR and Sanger sequencing revealed that the P2A-mCherry fragment was successfully inserted between exon 4 and the 3′ untranslated region of CRX in the BC1 (CRX-wild-type [WT]) hiPSCs (Figures S1B and S1C).

The edited hiPSCs exhibited a flat, dense, and smooth morphology typical of hiPSCs (Figure S1D) and maintained the normal male 46XY karyotype (Figure S1E). Immunofluorescence (IF) staining analysis confirmed that they expressed pluripotent markers (Figure S1F). Quantitative reverse-transcription PCR (RT-qPCR) analysis also indicated that, compared with CRX-WT hiPSCs, the CRX-mCherry hiPSCs showed no significant differences in the mRNA levels of several pluripotent marker genes (Figure S1G). Additionally, we performed teratoma formation assay in NOD/SCID mice, and the results demonstrated that the CRX-mCherry hiPSCs could differentiate into tissues representing all three germ layers: pigmented epithelial tissue of the ectoderm, adipose tissue of the mesoderm, and glandular tissue of the endoderm (Figures S1H–S1J). These results confirm the successful generation of CRX-mCherry reporter hiPSCs.

Tracking of mCherry+ cells localization during RO development

To assess whether the CRX-mCherry reporter hiPSCs could perform the expected function, they were differentiated into ROs step by step, with our previously established methods (Figures 1A and S3A–S3D). We first observed weak mCherry fluorescence at the outer edge of the RO at D44 (Figure S3E). As differentiation progressed, the distribution of mCherry gradually shifted from the outer to the inner part of the RO, with the signal intensity increasing over time. By day 50 (D50), we could easily distinguish the mCherry fluorescence under the microscope. The signal intensity and density further increased by D65 and continued to rise until D120. Eventually, by D160, the entire RO was filled with mCherry fluorescence (Figures 1A and 1B).

Figure 1.

Figure 1

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Tracking of mCherry+ cell localization during RO development

(A) Schematic of the RO differentiation protocol and expression pattern of CRX. EBs, embryonic bodies; EFs, eye fields; NR, neural retina; RPE, retinal pigment epithelium; PR, photoreceptor cells; RDM, retinal differentiation medium; RC1, retinal culture medium 1; RC2, retinal culture medium 2.

(B) Fluorescence imaging of mCherry expression in CRX-mCherry ROs from early to late developmental stages. BF, bright field; scale bar, 100 μm.

(C) IF staining was performed to assess the localization and expression of CRX relative to mCherry. Scale bar, 100 μm.

To further investigate whether mCherry could effectively monitor the expression and localization of CRX, we performed IF staining and confirmed that mCherry and CRX were indeed co-localized at various stages of ROs (Figure 1C). This indicates that the CRX-mCherry reporter RO system was successfully generated and could be applied in the downstream research.

mCherry specifically labels pan-photoreceptor cells during RO development

CRX is recognized as a marker that labels photoreceptor cells throughout their development from early to late stages (Furukawa et al., 1997). To evaluate whether mCherry in the RO could monitor the development of photoreceptor cells, we conducted IF staining from D65 to D120, mCherry, representing CRX, consistently co-localized with pan-photoreceptor cell marker RCVRN (Figure S4A), indicating that mCherry effectively labels photoreceptor cells. Similarly, mCherry also co-localized with cone precursor cell marker RXRγ (Figure S4B); however, the ratio of co-localized cells gradually decreased as development progressed. This decline may be attributed to the gradual specification of rod photoreceptor cells.

Furthermore, we did not observe mCherry expression in retinal pigment epithelium cells (RPEs) (Figure S3F) or other retinal cell types, including VSX2+ retinal progenitor cells (RPCs), HuD+ retinal ganglion cells (RGCs) and amacrine cells (ACs), SOX9+ Müller glia cells (MGs), or VSX2+ bipolar cells (BCs) (Figures S5A–S5D), nor did it induce abnormal proliferation of photoreceptor cells (Figure S5E). This suggests that mCherry labeling does not affect the post-mitotic cell cycle of photoreceptor cells. Thus, the CRX-mCherry real-time traceable reporting fluorescent RO system was successfully established.

Combined CUT&Tag-seq and RNA-seq analysis reveals the profile of CRX target genes

To examine the function of target genes regulated by CRX, we performed CUT&Tag-seq analysis in groups, one group included two samples without CRX antibody treatment (−Ab), and the other group had three samples with CRX antibody treatment (+Ab). Principal-component analysis (PCA) was employed to differentiate the correlation among the samples, clearly distinguishing between the −Ab and +Ab groups (Figure S6A). We identified CRX-bound regions (CBRs) through peak calling across the whole genome, comparing the +Ab to the −Ab group. In +Ab group, most of these peaks were in distal intergenic regions (46.53%) and introns (42.71%), with a smaller proportion found in promoter regions (6.72%). Additionally, a limited number of peaks were detected in the UTR (2.63%), exons (0.73%), and downstream regions (0.67%) (Figures 2A, 2B, and S6C), while very few peaks were observed in the −Ab control group (Figures S6B and S6C). Differential volcano plots indicated that 2,772 peaks (related to 1,954 genes) were upregulated, two peaks (related to two genes) were downregulated, and 830 peaks exhibited no significant difference in the +Ab group relative to the −Ab group (Figure 2C).

Figure 2.

Figure 2

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Identification of CRX-regulated target genes using CUT&Tag-seq

(A) Distribution of reads associated with CRX-bound regions (CBRs) on chromosomes.

(B) Distribution of CBRs across genome-wide functional elements in the CRX antibody group (+Ab).

(C) Volcano plot displaying differentially enriched peaks genome-wide in the CRX antibody group (+Ab) compared with the no-antibody control group (−Ab). −Ab, without CRX antibody; +Ab, with CRX antibody. Each dot represents a peak; red, green, and black dots indicate upregulated, downregulated, and non-significant peaks, respectively.

(D) Motif enrichment analysis of CBRs. Unerased E-value refers to the E-value of the motifs calculated without erasing the sites of previously identified motifs.

(E) Network of CRX-regulated downstream transcription factors (TFs) inferred from CUT&Tag-seq data. Nodes represent TFs, and edges represent regulatory relationships.

(F–M) CRX binding signals on cone photoreceptor-specific TF RXRG (F), rod photoreceptor-specific TF NRL (G), retinal progenitor cell (RPC)-specific TFs SIX3 and MEIS1 (H and I), amacrine cell (AC)-specific TFs TFAP2C and EBF3 (J and K), retinal ganglion cell (RGC)-specific TF POU6F2 (L), and horizontal cell (HC)-specific TF PROX1 (M).

MEME-ChIP analysis identified two motifs within the CBRs that closely resemble those of mouse Crx (motif: TAATCC) (Figure 2D), likely attributable to the conserved homeodomain regions of human CRX and mouse Crx (Figure S9A). Our analysis revealed that CRX regulates 155 TFs (Figure 2E), containing TFs specifically expressed in photoreceptors and other retinal cell subtypes. Interestingly, we found that CRX could also regulate its own expression (Figures 2E, 5A, and S7A), a phenomenon similarly observed in mice (Corbo et al., 2010; Furukawa et al., 2002), suggesting this regulatory mechanism is highly conserved across species.

Figure 5.

Figure 5

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Comparative analysis of target genes between human CRX and mouse Crx

(A) Venn diagram illustrating overlapping CRX target genes between our CUT&Tag-seq data and previously published ChIP-seq datasets.

(B) Venn diagram analysis revealing the intersection between Crx target genes in mouse ChIP-seq data and genes that are upregulated or downregulated in Crx−/− mice relative to wild-type mice in the microarray dataset.

(C and E) Venn diagram analysis revealing the gene intersections among four omics datasets, where the ChIP-seq and microarray data were derived from Corbo et al. (2010) and Hsiau et al. (2007), respectively.

(D and F) Heatmaps showing the genes in the common intersection of the four omics datasets derived from (C) and (E). Genes marked in red indicate those reported to cause IRDs. mN, mCherry negative; mP, mCherry positive.

ROs were dissociated and flow-sorted into mCherry+ (37.8% mCherry+ cells of total, data not shown) and mCherry− cells for RNA sequencing (RNA-seq). Pearson sample correlation analysis revealed good intra-group reproducibility (Figure S8A). The resulting volcano plot revealed 4,200 differentially expressed genes (DEGs), with 1,256 DEGs upregulated and 2,944 DEGs downregulated in the mCherry+ group compared with the mCherry− group (Figure 3A). The mCherry+ cells displayed a comprehensive photoreceptor cell signature, characterized by the expression of pan-photoreceptor markers such as CRX, OTX2, PRDM1, and RCVRN (Figure 3C); cone photoreceptor-specific markers including RXRG, THRB, ARR3, PDE6H, and PDE6B (Figure 3C); as well as rod photoreceptor-specific markers such as NRL and NR2E3 (Figure 3C). The mCherry− cells displayed a transcriptomic profile characteristic of RPC (VSX2+, CCND1+, MEIS1+, SIX3+) (Figure 3C), RGC (POU4F2+, POU6F2+, ATOH7+) (Figure 3C), AC (TFAP2C+, EBF3+) (Figure 3C), horizontal cell (HC) (PROX1+, ONECUT1+, ONECUT2+) (Figure 3C), and MG (SOX9+, RLBP1+) (Figure 3C). CUT&Tag-seq analysis revealed that several of these genes are directly regulated by CRX, including the cone-specific TFs RXRG, THRB (Figures 2E, 2F, 3C, and S7B), and the rod-specific TFs NRL and NR2E3 (Figures 2E, 2G, 3C, and S7C), and pan-photoreceptor TF PRDM1 (Figures 2E, 3C, and S7D).

Figure 3.

Figure 3

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Combined CUT&Tag-seq and RNA-seq analysis reveals the profile of CRX target genes

(A) Volcano plot illustrates the differentially expressed genes (DEGs) between mCherry+ and mCherry− cells from RNA-seq data. mN, mCherry negative; mP, mCherry positive.

(B) Venn diagram comparing CRX target genes identified by CUT&Tag-seq and DEGs from RNA-seq (A). There are 386 genes common to both CUT&Tag-seq and RNA-seq that are potentially repressed by CRX, while 333 genes are potentially activated by CRX.

(C) Heatmap of DEGs corresponding to retinal cell type-specific marker genes in RNA-seq data. The red-labeled genes indicate direct targets of CRX in the CUT&Tag-seq data. mN, mCherry negative; mP, mCherry positive; PR, photoreceptor cell; RGC, retinal ganglion cell; AC, amacrine cell; HC, horizontal cell; MG, Müller glia cell; RPC, retinal progenitor cell.

(D and E) Top five enriched Gene Ontology (GO) terms of common DEGs identified by RNA-seq and CUT&Tag-seq. CC, cellular component; MF, molecular function; BP, biological process.

Moreover, CRX can function as a transcriptional repressor as well, downregulating the expression of specific non-photoreceptor retinal cell-associated TFs to preserve the expression patterns of photoreceptor cells, such as the RPC-specific markers SIX3 and MEIS1 (Heine et al., 2008) (Figures 2E, 2H, 2I, and 3C), the AC-specific markers TFAP2C and EBF3 (Bassett et al., 2012; Watanabe et al., 2015) (Figures 2E, 2J, 2K, and 3C), the RGC-specific marker POU6F2 (Sapkota et al., 2014) (Figures 2E, 2L, and 3C), and the HC-specific marker PROX1 (Figures 2E, 2M, and 3C). Furthermore, CRX also regulates additional TFs whose retinal expression patterns have not been fully characterized, including genes exhibiting both high and low expression levels in photoreceptor cells (Figure S8B).

To further investigate the function of CRX in the development of photoreceptor cells, we conducted a comprehensive analysis by integrating CUT&Tag-seq and RNA-seq data. Venn diagrams were utilized to depict the target genes associated with CRX as identified in the CUT&Tag-seq data, in conjunction with the DEGs that were found in the RNA-seq analysis. This approach revealed an intersection of 333 genes that exhibited upregulation and 386 genes that demonstrated downregulation (Figure 3B). Furthermore, Gene Ontology (GO) enrichment analysis of these genes indicated that the upregulated genes were primarily associated with cilia development, the formation of inner and outer segments, light stimulation, and phototransduction pathways related to photoreceptor cells (Figure 3D). Conversely, the downregulated genes were predominantly linked to functions specific to other retinal cell types, including cell junctions, synapses, adhesion, and ion binding (Figure 3E).

These findings suggest that human CRX plays a pivotal role in the development of photoreceptor cells by not only enhancing the expression of genes critical for the structure and metabolism of photoreceptor cells but also by acting as a transcriptional repressor to inhibit the expression of non-photoreceptor retinal genes, thereby facilitating the specialization and maturation of photoreceptor cells.

CRX controls photoreceptor cells development by binding to promoters of target genes

Promoter is one of the most active regions in gene regulation. To further elucidate the function of CRX in the regulation of target genes, we conducted an analysis of CBRs within promoter regions (< 2 kb) for a total of 347 peaks (related to 347 genes), utilizing CUT&Tag-seq data (Figure 4A). Among these genes, 35 were identified as upregulated, while 71 were downregulated based on RNA-seq data (Figure 4B). Notably, several genes, including CPLX4, a gene associated with synaptic vesicles, have been previously documented as being regulated by both Crx and Nrl in murine models (Assawachananont et al., 2018). Additionally, ARR3, a gene specific to cone photoreceptor cells, has also been reported to be under the regulation of Crx (Chen et al., 1997). Our findings support these previous observations, confirming that these genes are indeed regulated by CRX (Figures 4C and 4D).

Figure 4.

Figure 4

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CRX controls photoreceptor development by binding to cis-regulatory elements in the promoters of target genes

(A) Volcano plot of differentially CRX-bound peaks in promoter regions (within 2 kb upstream of the TSS), comparing samples with (+Ab) and without (−Ab) CRX antibody. Each dot represents a peak, 347 peaks correspond to 347 genes. Red, green, and black dots indicate upregulated, downregulated, and non-significant peaks, respectively. −Ab, without CRX antibody; +Ab, with CRX antibody.

(B) Venn diagram comparing genes identified by CUT&Tag-seq (A) and differentially expressed genes (DEGs) from RNA-seq. Thirty-five genes common to both CUT&Tag-seq and RNA-seq are potentially repressed by CRX, while 71 genes are potentially activated by CRX.

(C and D) Peak distribution of two genes reported to be regulated by CRX. The black arrows indicate the transcription direction of each gene.

(E) Heatmap of gene expression in mCherry+ and mCherry− groups, as determined by RNA-seq. mN, mCherry negative; mP, mCherry positive.

(F–J) Peak distribution of the common DEGs from (B), black arrows indicate the direction of transcription.

(K and L) Schematic representation of CRX motif 1 and motif 2 in the promoters of RP1L1 and PCDH8. +1 represents the transcription start site.

(M–Q) Relative luciferase activity of promoters regulated by CRX. Data are presented as the ratio of firefly to Renilla luciferase activity. n = 4 biological replicates per group; error bars represent the mean ± SD. One-way ANOVA was used for statistical significance analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001; ns, p > 0.05.

Furthermore, we identified several novel genes that have not been well characterized previously, such as GNB3, IGSF21, and RP1L1, which were expressed in mCherry+ photoreceptor cells but not in mCherry− cells (Figures 4B and 4E). In contrast, genes like SOX4 and PCDH8 were expressed in mCherry− cells but absent in mCherry+ cells (Figures 4B and 4E). Importantly, these genes displayed strong CRX binding signals within their promoter regions (Figures 4F–4J). We also identified multiple instances of CRX motif 1 and motif 2 elements within the promoters of these genes (Figures 4K, 4L, and S9B–S9D). To validate the regulatory role of CRX on these genes, we performed a dual-luciferase reporter gene assay in HEK293T cells. Our results indicate that the promoter activity of RP1L1, a gene associated with IRDs and expressed in photoreceptor cilia (Liu et al., 2024; Noel and MacDonald, 2020), was significantly upregulated by CRX, this regulatory response was dependent on the dose of CRX (Figure 4O). Moreover, our findings indicate that CRX also regulates the ciliary axis, which encompasses RP1 and PRPH2 (Figures S8C and S8D). Conversely, PCDH8, previously thought to be expressed in ganglion cell layer (GCL) and inner nuclear layer (INL) of developing vertebrate retina (Etzrodt et al., 2009), was found to be significantly downregulated by CRX (Figure 4Q). Unfortunately, some genes identified as CRX target genes cannot be regulated or respond to CRX in the HEK293T system (Figures 4M, 4N, and 4P).

Collectively, these findings suggest that CRX plays a critical role in regulating the expression of both photoreceptor-related and unrelated genes by binding to their promoters. These experiments highlight the dual functionality of CRX in the development and maintenance of photoreceptor cells.

Comparative analysis of target genes between human CRX and mouse Crx

To explore the conservation and divergence of CRX-regulated target genes among different species, we compared CRX target genes between our human CUT&Tag-seq data and mouse ChIP-seq data (Corbo et al., 2010), which revealed species- and developmental stage-specific differences. Since the mouse ChIP-seq data were derived from adult retinas, Crx predominantly bound genes associated with mature photoreceptor phototransduction function and maintenance, such as Rho, Opn1sw, Opn1mw, and Gnat1 (Figure 5A). In contrast, human CRX targets in our mid-developmental stage ROs were enriched for genes involved in photoreceptor early development, such as RXRG, ARR3, and PRDM1 (Figure 5A), mutations in which are associated with early-onset retinal abnormalities. The overlapping genes between the two species datasets, including CRX, THRB, NRL, and NR2E3 (Figure 5A), likely represent core targets essential for both photoreceptor development and functional maintenance. Interestingly, we found that the gene RP1L1, which can be upregulated by CRX in human ROs and HEK293T cells, is also regulated by Crx in mice (Figure 5A).

To further investigate whether murine Crx can bidirectionally regulate target genes in the retina, we integrated the analysis of Crx ChIP-seq and Crx−/− microarray datasets (Hsiau et al., 2007) in mice and found that Crx indeed possesses such capability (Figure 5B). Furthermore, Venn diagrams identified a set of genes that are bidirectionally regulated by CRX and highly conserved between humans and mice, as well as across developmental and adult stages (Figures 5C–5F).

Notably, our findings reveal that RCVRN, a pan-marker gene that exhibits sustained high expression during photoreceptor development and maintenance in both human and mouse retinas, is regulated in mice but lacks detectable CRX binding signals in human retinal tissues (Figures 5A and 5B). Moreover, in our studies on human ROs, GNB3 and IGSF21 have been identified as potential targets of positive regulation by CRX (Figures 4B, 4F, and 4G). However, in mice, these two genes appear to be repressed by Crx (Figure 5B). This species-specific difference likely reflects subtle divergences in CRX regulatory networks between humans and mice, notwithstanding their broadly conserved functions.

CRX knockout leads to aberrant PROX1 expression in photoreceptor cells

Gene knockout is one of the most effective approaches to elucidate gene function. To investigate the regulatory role of CRX on its target genes, we employed CRISPR-Cas9 technology to generate CRX knockout (CRX-KO) hiPSCs. Specifically, we designed a single guide RNA (sgRNA) targeting exon 2 of CRX, which is proximal to the start codon (Figure 6A). This sgRNA, along with Cas9, was delivered via a single plasmid into CRX-WT hiPSCs. Following puromycin selection and validation by PCR (data not shown) and Sanger sequencing (Figure 6A), two homozygous CRX-KO hiPSC lines were successfully established, harboring deletions of 11 bp and 2 bp (Figures 6A and S10A), respectively.

Figure 6.

Figure 6

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CRX knockout leads to aberrant PROX1 expression in photoreceptor cells

(A) Schematic illustration of the construction process for CRX-KO hiPSC lines using CRISPR-Cas9 technology, with Sanger sequencing confirming CRX knockout in hiPSCs. The red boxes indicate the positions of the start codon. NHEJ, non-homologous end-joining; CRX-KO-VF, CRX knockout validation forward primer; CRX-KO-VR, CRX knockout validation reverse primer.

(B and C) Western blot analysis of CRX, OTX2, and RCVRN expression in CRX-WT and CRX-KO D65 ROs.

(D and E) Statistical analysis of western blot results in (C). n = 4 biological replicates for each group containing five ROs; error bars represent the mean ± SD. A two-tailed Student’s t test was used for statistical significance analysis. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(F and G) IF staining of PROX1 and RXRγ in CRX-WT and CRX-KO D120 ROs. Scale bar, 100 μm.

(H) Schematic illustration of the mechanism through which CRX modulates photoreceptor cell development. CRX precisely controls photoreceptor cell development and maintenance by promoting photoreceptor-specific transcription factors (TFs) while suppressing non-photoreceptor TFs. RPC, retinal progenitor cell; RGC, retinal ganglion cell; AC, amacrine cell; HC, horizontal cell; PR, photoreceptor cell.

These CRX-KO hiPSCs were subsequently differentiated into ROs (Figure S10B). To characterize the functional phenotypes of CRX-KO ROs, we performed western blot analysis, which confirmed the complete loss of CRX protein in CRX-KO ROs (Figure 6B), along with significant downregulation of the photoreceptor cell markers RCVRN and OTX2 (Figures 6C–6E). IF staining results further confirmed the downregulated expression of RCVRN and OTX2 (Figures S11A–S11D). The IF staining also verified the loss of expression of both cone photoreceptor maturation markers ARR3 and L/M opsin (Figures S12A, S12C, S12E, S12G, S12I, and S12K) and the rod photoreceptor maturation marker Rhodopsin (Figures S12J and S12L), while not affecting the expression of cone-specific TF RXRγ and rod-specific TF NRL (Figures S11C, S12B, S12D, S12F, and S12H).

As described above, we identified several TFs involved in non-photoreceptor cells development as potential CRX targets, which are normally suppressed in photoreceptor cells (Figures 2E, 2H–2M, 3C,and S8B). To validate these findings, we conducted IF colocalization experiments of PROX1 (an HC marker) and RXRγ (a cone photoreceptor cell marker) in CRX-WT and CRX-KO ROs. The results revealed colocalization of PROX1 and RXRγ in CRX-KO ROs (Figure 6G), contrasting with their distinct expression patterns in CRX-WT ROs (Figure 6F). Since RXRγ labels RGC in mouse retinas (Lyu and Mu, 2021), we performed additional staining with the RGC-specific marker RBPMS to confirm this possibility. Our results demonstrated that there was some colocalization between RXRγ and RBPMS (Figure S13A). Furthermore, we identified a subset of cells with weak RXRγ expression that were OTX2-negative (Figure S11C). These indicate that in the human retina, RXRγ is weakly expressed in a small subset of RGCs beyond photoreceptor cells.

To further verify that aberrant PROX1 expression occurs in photoreceptor cells, we subsequently performed IF colocalization staining using the pan-photoreceptor marker RCVRN and PROX1. The results confirmed aberrant PROX1 expression was located in RCVRN+ photoreceptor cells (Figure S13B).

Altogether, these findings indicate that CRX knockout leads to the ectopic expression of PROX1 in photoreceptor cells, validating that CRX can function as a transcriptional repressor for non-photoreceptor cells development-related genes, beyond its well-established role in cone and rod photoreceptor cells specification (Hsiau et al., 2007; Peng et al., 2005; Pittler et al., 2004) (Figure 6H).

Discussion

To dissect the transcriptional regulatory network of human CRX in photoreceptor development and generate high-quality profiles of both CRX-mediated chromatin-binding and gene expression, we established a CRISPR-Cas9-edited CRX-mCherry fluorescent reporter hiPSC line and differentiated it into ROs. This model was pivotal for acquiring paired RNA-seq and CUT&Tag-seq datasets: mCherry enabled real-time tracking of CRX expression dynamics across RO development and facilitated FACS-based isolation of pure photoreceptor populations, ensuring the cellular homogeneity critical for reliable transcriptional (RNA-seq) and chromatin-binding (CUT&Tag-seq) analyses. Leveraging this validated system, we generated the first CRX-bound CUT&Tag-seq dataset from human retinal tissues, and by integrating it with photoreceptor-specific RNA-seq data, established the first comprehensive repertoire of human CRX-regulated targets. These integrated datasets not only filled the gap in human-specific CRX chromatin-binding and transcriptional regulatory information but also provided a robust foundation for subsequent functional validation, linking CRX binding events to tangible gene expression outcomes.

Through CUT&Tag-seq analysis, we identified two CRX-bound motifs in human retinal tissues that exhibit conservation with those observed in mice, contrasting with previous findings from animal model studies using ChIP-seq. This conservation likely stems from the complete identity of the HD domain (a main part of DBD) between human CRX and animal counterparts (Figure S9A), while observed differences may be attributed to divergence in the TAD and other regions of the DBD. Significantly, we established the first comprehensive profile of CRX-regulated target genes and associated pathways in human retinal tissues, providing a valuable reference for future investigations into CRX-related retinal diseases. Our dual-luciferase assay revealed RP1L1 and PCDH8 as novel direct targets of CRX, underscoring its dual role in transcriptional activation and repression (promoting RP1L1 expression while inhibiting PCDH8). Notably, RP1L1, which shares homology with RP1, has been implicated in IRDs. These findings offer mechanistic insights into how CRX mutations can lead to a spectrum of overlapping yet distinct clinical phenotypes, including LCA, RP, and CoRD. The observed phenotypic variability likely reflects differential regulation of target genes by CRX, leading to diverse pathogenic outcomes.

Moreover, our investigation revealed that CRX exerts inhibitory effects on several cell-specific transcriptional factors associated with non-photoreceptor lineages. Through the generation of CRX-KO hiPSCs and ROs, we observed distinct regulatory patterns: in CRX-WT ROs, CRX effectively suppressed the expression of PROX1, a marker for HC, while this suppression was markedly attenuated in CRX-KO ROs. Intriguingly, Prox1 was found to be upregulated across various mouse photoreceptor injury models (Lee et al., 2025), implying its potential role as a stress-responsive factor in retina self-protection. These findings highlight the crucial role of human CRX in repressing retinal cell type-specific genes, a mechanism essential for proper photoreceptor cell development and maintenance. While previous studies have established the pivotal role of CRX in determining cone and rod photoreceptor cell fates through the activation and repression of key photoreceptor genes (Hsiau et al., 2007; Peng et al., 2005; Pittler et al., 2004; White et al., 2016), our study provides the first experimental evidence demonstrating that CRX plays a crucial role in maintaining photoreceptor cell identity through the transcriptional repression of non-photoreceptor lineage genes.

Our CUT&Tag-seq analysis identified striking differences in the genomic binding patterns between human CRX and mouse Crx. For instance, regarding core TFs, specifically, mouse Crx primarily targets the promoter regions of core photoreceptor TFs such as Nrl and Nr2e3 to drive transcriptional activation (Corbo et al., 2010), whereas human CRX exhibits numerous binding sites only in non-canonical regulatory regions, including core TFs such as PRDM1, RXRG, THRB, NRL, and NR2E3. The canonical role of TFs is to bind to the promoters of target genes, recruit RNA polymerase II, and directly determine whether transcription of the gene is initiated (Latchman, 1997; Roeder, 1996). While CRX-mediated regulation involves enhancer activation and chromatin remodeling (Ruzycki et al., 2018), we speculate that human CRX may recruit histone acetyltransferases (HATs) or chromatin remodelers to intronic or exonic regions, thereby modulating the accessibility of distal regulatory elements or exon-exon junctions to shape gene expression. This mechanism explains the correlation between CRX binding to non-promoter regions and the upregulation of target TFs observed in our RNA-seq data. Importantly, this regional specificity likely enables nuanced control of photoreceptor gene networks through enhancer activation, chromatin remodeling, and potential post-transcriptional regulation—a mechanism that has also been reported for other TFs such as TP53 and CMYC (Wojtyś and Oroń, 2023)—highlighting the importance of studying human models to understand human retinal development and disease.

While our study advances understanding of human CRX regulation, there exist several limitations. First, although our analysis suggested potential CRX regulation of GNB3, IGSF21, and SOX4, subsequent dual-luciferase assays failed to detect corresponding regulatory responses even with increasing CRX dosage, likely due to the use of non-physiological HEK293T cells, which lack the native co-factor pool and chromatin context required for CRX function, such as coactivators like CBP, P300, NRL, and NR2E3 (Corbo et al., 2010; Hennig et al., 2013; Peng et al., 2005; Peng and Chen, 2007; Sun and Chen, 2023). Second, we lack validation of the causal role of intronic or exonic CRX binding sites in gene regulation (e.g., via mutagenesis experiments). Therefore, further experiments are required to clarify and validate the distinct functions of CBRs in different genomic regions.

In summary, our study elucidates the dual regulatory role of human CRX in photoreceptor development through a combination of advanced genomic and cellular techniques. Notably, our work represents the first to generate CRX-bound CUT&Tag-seq profile from human retinal tissue, characterize the regulatory network of human CRX, and perform cross-species comparison of CRX target genes between humans and mice, uncovering several differences in CRX-mediated regulatory mechanisms across these species. These findings not only deepen our understanding of the transcriptional mechanisms of CRX but also provide critical insights into the molecular basis of CRX-associated retinal degenerative diseases. More validation will be needed to further verify the CRX target genes revealed in this study. Future studies should explore the functional significance of CRX binding in non-canonical regulatory regions and investigate its interactions with co-factors to fully unravel the complexities of its regulatory network. This work lays a foundation for developing targeted therapeutic strategies for retinal disorders linked to CRX dysfunction.

Methods

Generation of CRX-mCherry reporter and CRX-KO hiPSC line

BC1 (CRX-WT) hiPSCs (Chou et al., 2011) were used as parental cells for generating CRX-mCherry and CRX-KO hiPSC lines. Plasmid construction and electroporation were performed following the protocol we previously described (Guan et al., 2022); for more details, see the supplementary methods.

Cell culture and RO differentiation

All hiPSCs were maintained in mTeSR1 (STEMCELL) medium, with daily medium changes. The cells were passaged every 5 days using 0.5 mM EDTA (Invitrogen) for enzymatic digestion. RO differentiation was performed according to the protocol we previously established (Guan et al., 2021; Zhong et al., 2014).

CUT&Tag-seq analysis

The CRX-mCherry RO library was constructed using the Hyperactive Universal CUT&Tag Assay Kit (Vazyme, TD903) according to the manufacturer’s instructions. Briefly, 10 D90 ROs were lysed into single cells using papain (Worthington Biochemical) at 37°C for 30 min, then incubated with ConA beads at room temperature for 10 min, followed by an overnight incubation with the primary antibody at 4°C. The cells were divided into two groups: one group was treated with the CRX antibody (1:50), while the other group served as a control and did not receive the antibody. The secondary antibody (1:100) was incubated at room temperature for 1 h, followed by a 1-h incubation with Hyperactive pA/G-Transposon, also at room temperature. Finally, TruePrep Tagment Buffer L (TTBL) was added to fragment the DNA. Subsequently, the DNA libraries were amplified using TruePrep Index Kit (V2 for Illumina) (Vazyme, TD202) according to the manufacturer’s instructions.

Quality control of the library prepared through the CUT&Tag experiment was conducted using the Qubit3.0 (ThermoFisher) and Agilent Bioanalyzer 2100, followed by sequencing on the Novoseq 6000 sequencer. The clean reads were aligned to the reference genome using Bowtie2 alignment software (Langmead and Salzberg, 2012), filtering out reads with low alignment quality values. Only reads that aligned uniquely to the genome were utilized for subsequent analysis. Peak calling analysis was performed across the entire genome using MACS2 analysis software (Zhang et al., 2008), with a threshold set at false discovery rate (FDR) <0.05. The ChIPseeker R package (Yu et al., 2015) was used to perform genome-wide element annotation on the peaks, the peak location information on the genome and the sequence information of the peak regions were analyzed to filter out peak-related genes. (Genes with peaks located within 2 kb upstream of the transcription start sites (TSSs), the gene body, and 2 kb downstream of the transcription termination sites (TTSs) were defined as peak-associated genes.) Subsequently, differential peak analysis was performed using the DiffBind software (Stark and Brown, 2011), with the screening criteria of |log2FC| ≥ 1 and FDR ≤0.05 to identify differential peaks. Visualization of the peaks was accomplished using the Integrative Genomics Viewer (IGV) software (Robinson et al., 2011). Motif analysis was conducted using the MEME Suite (http://meme-suite.org/), employing MEME-ChIP to detect significant motif sequences within the peak sequences. In the GO enrichment analysis of peak-related genes, entries with FDR <0.05 were identified as significantly enriched. Sequencing and analysis were completed by Gene Denovo Biotechnology Co., Ltd (Guangzhou, China).

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Xiufeng Zhong (zhongxf7@mail.sysu.edu.cn).

Materials availability

The plasmids and cell lines generated in this study will be provided upon request.

Data and code availability

The raw and processed omics data from RNA-seq and CUT&Tag-seq have been deposited in the GEO database (https://www.ncbi.nlm.nih.gov/geo/), with the GEO accession number GSE290304.

Acknowledgments

We thank professor Linzhao Cheng from the University of Science and Technology of China for providing the normal BC1 (CRX-WT) hiPSCs. This research was funded by the National Key R&D Program Project of the Ministry of Science and Technology (2023YFC2506100), the National Natural Science Foundation of China (82172957, 81970842), Guangdong Basic and Applied Basic Research Foundation (2022A1515110631), and Guangdong Basic Research Center of Excellence for Major Blinding Eye Diseases Prevention and Treatment (2024-PIZC-029).

Author contributions

Y.W., B.X., X.S., G.G., Y.G., D.Z., and P.X. conducted the experiments. X.Z., Y.W., B.X., and X.S. collaborated on experimental design and data analysis. X.Z. and Y.W. wrote the manuscript. X.Z. also secured funding and provided final supervision.

Declaration of interests

The authors declare no competing interests.

Published: December 18, 2025

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2025.102747.

Supplemental information

Document S1. Figures S1–S13, Tables S1–S3, and supplementary methods
mmc1.pdf (3.2MB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (22.9MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S13, Tables S1–S3, and supplementary methods
mmc1.pdf (3.2MB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (22.9MB, pdf)

Data Availability Statement

The raw and processed omics data from RNA-seq and CUT&Tag-seq have been deposited in the GEO database (https://www.ncbi.nlm.nih.gov/geo/), with the GEO accession number GSE290304.

Articles from Stem Cell Reports are provided here courtesy of Elsevier

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