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Targeting specific DNA G-quadruplexes with CRISPR-guided G-quadruplex-binding proteins and ligands

Nature Cell Biology volume 26, pages 1212–1224 (2024)Cite this article

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Abstract

Despite the demonstrated importance of DNA G-quadruplexes (G4s) in health and disease, technologies to readily manipulate specific G4 folding for functional analysis and therapeutic purposes are lacking. Here we employ G4-stabilizing protein/ligand in conjunction with CRISPR to selectively facilitate single or multiple targeted G4 folding within specific genomic loci. We demonstrate that fusion of nucleolin with a catalytically inactive Cas9 can specifically stabilize G4s in the promoter of oncogene MYC and muscle-associated gene Itga7 as well as telomere G4s, leading to cell proliferation arrest, inhibition of myoblast differentiation and cell senescence, respectively. Furthermore, CRISPR can confer intra-G4 selectivity to G4-binding compounds pyridodicarboxamide and pyridostatin. Compared with traditional G4 ligands, CRISPR-guided biotin-conjugated pyridodicarboxamide enables a more precise investigation into the biological functionality of de novo G4s. Our study provides insights that will enhance understanding of G4 functions and therapeutic interventions.

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Fig. 1: Targeted G4s in specific genome loci using dCas9–nucleolin.
Fig. 2: MycG4 is selectively stabilized by dCas9–nucleolin.
Fig. 3: Targeting stabilization of telomere G4s disrupts telomere homeostasis via dCas9–nucleolin.
Fig. 4: Itga7 promoter G4 stabilized by dCas9–nucleolin inhibits muscle cell differentiation.
Fig. 5: dCas9–mSA/Bio–PDC stabilizes G4 in targeted genome loci.
Fig. 6: Selectively stabilizing de novo G4 by dCas9–SunTag–mSA-guided Bio–PDC.
Fig. 7: dCas9–SunTag–mSA/Bio–PDC selectively stabilizes NEAT1 and MALAT1 G4s.

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Data availability

The ATAC-seq and RNA-seq data generated from this study have been deposited in the Gene Expression Omnibus (GSE255625). The following previously published datasets were used: hg38 (https://www.ensembl.org/Homo_sapiens/Info/Index), PDS and PhenDC3 Chem-map (GSE209713)23, BG4 CUT&Tag (GSE181373)22, ATAC-seq (GSE162299)24. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank S. Balasubramanian and M. Farrow for their help on the synthesis. Financial support was provided by the National Key R&D Program of China (2019YFA0709202 to X.Q.; 2021YFF1200700 to J.R.; 2022YFA1205804 to C.Z.), the National Natural Science Foundation of China (91856205 and 21820102009 to X.Q.; 22237006 to J.R.; 22107098 to G.Q.; 22122704 to C.Z.) and Jilin Innovation Project (2023DJ02 to X.Q.).

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  1. These authors contributed equally: Geng Qin, Zhenqi Liu.

Authors and Affiliations

  1. Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, P. R. China

    Geng Qin, Zhenqi Liu, Jie Yang, Xiaofeng Liao, Chuanqi Zhao, Jinsong Ren & Xiaogang Qu

  2. University of Science and Technology of China, Hefei, P. R. China

    Geng Qin, Zhenqi Liu, Jie Yang, Xiaofeng Liao, Chuanqi Zhao, Jinsong Ren & Xiaogang Qu

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Contributions

X.Q. was the principal investigator who conceived and designed the study, obtained financial supports and approved the final version of the paper. J.R. was the other principal investigator who conceived the study. G.Q. and Z.L. performed the experiments; G.Q., Z.L., J.Y., X.L. and C.Z. conducted data management; G.Q. performed the statistical analyses, interpreted the results and drafted the paper. All the authors read and approved the final version of the paper.

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Correspondence to Xiaogang Qu.

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Extended data

Extended Data Fig. 1 Targeted G4s in specific oncogene promoter region using dCas9–nucleolin.

a, The proposed model of MYC transcriptional regulation by MycG4 and nucleolin. b, Binding curves of nucleolin with MycG4 and a hairpin as the example of non-G4 structure determined by an adapted enzyme linked immunosorbent assay (ELISA). c, The human MYC locus. MycG4 site is indicated by a yellow box. The locations of the targets (3–6) for sgRNAs (sgR-3-6) are indicated by red bars. d, Binding curves of nucleolin with KRAS G4 and VEGFA G4 determined by ELISA. e, The human KRAS locus. KRAS G4 site is indicated by a yellow box. The locations of the targets (1–4) for sgRNAs (sgR-KRAS-1-4) are indicated by red bars. f, Inhibition of EGFP protein expression in HEK293T cells by sgR-KRAS-1-4, each expressed with or without dCas9–nucleolin. EGFP reporter gene was downstream of the KRAS promoter with or without KRAS G4. g, The human VEGFA locus. VEGFA G4 site is indicated by a yellow box. The locations of the targets (1–3) for sgRNAs (sgR-VEGFA-1-4) are indicated by red bars. h, Inhibition of EGFP protein expression in HEK293T cells by sgR-VEGFA-1-4, each expressed with or without dCas9–nucleolin. EGFP reporter gene was downstream of the VEGFA promoter with or without VEGFA G4. i, The mRNA levels of EGFP with the MycG4-WT or MycG4-Mut MYC promoter in the HEK293T cells transfected with dCas9–nucleolin and the indicated sgRNAs. j, The protein levels of dCas9 and dCas9–nucleolin in the HEK293T cells transfected with dCas9 or dCas9–nucleolin, detected by western blotting assays. Data are shown as mean ± SEM of three independent experiments, and P values were determined using two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in Source data.

Source data

Extended Data Fig. 2 dCas9–nucleolin targeting MycG4 inhibits MYC transcription and induces cell growth arrest.

a, The dCas9–nucleolin decreases the levels of both unprocessed and mature MYC RNA transcripts. Unprocessed MYC RNA transcript was measured using the intron specific primers. Mature MYC mRNA levels were measured using a pair of primer flanking intron 1. All RNA transcript levels were measured at desired time points after transfecting dCas9–nucleolin and sgR-5. b, The nucleolin/G4 complexes were captured by BG4 antibody through Anti-Flag Magnetic Beads, detected by western blotting assays. c, The proposed model of MYC transcriptional regulation by G4s, NM23H2, SP1, CNBP, hnRNP K and nucleolin. d-g, The occupancy of SP1, CNBP, hnRNP K and nucleolin in the promoter region of MYC was measured by ChIP in HeLa cells transfected with dCas9–nucleolin and sgR-5. h-k, The occupancy of SP1, CNBP, hnRNP K and nucleolin in the promoter region of MYC was measured by ChIP in HEK293T cells transfected with dCas9–nucleolin and sgR-5. l, Cells growth assays on HEK293T, HeLa, MCF-7 and K562 cells expressing dCas9–nucleolin and sgR-5. m, Genome browser tracks overlaid at HIF1A and NRAS genes. The dark green box highlights the G4 sites. Data are shown as mean ± SEM of three independent experiments, and P values were determined using two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in Source data.

Source data

Extended Data Fig. 3 Targeting telomere and Itga7 G4s by using dCas9–nucleolin.

a, Binding curves of nucleolin with telomere G4 determined by ELISA. b, Left: Immunofluorescence (IF) showing BG4 foci (red) in the nuclei of HeLa cells transfected with dCas9–nucleolin, sgR-Telo or sgR-Ctrl (n = 24). Scale bars, 5 μm. Right: The number of BG4 foci per nucleus. c, Left: IF showing BG4 foci (red) in the nuclei of HeLa cells transfected with dCas9 or dCas9–nucleolin (n = 30). Scale bars, 5 μm. Right: The number of BG4 foci per nucleus. d, The telomere length of the HeLa cells transfected with dCas9–nucleolin/sgR-Telo was detected by qRT-PCR. e, Binding curves of nucleolin with Itga7 G4 determined by ELISA. Data are shown as mean ± SEM of three independent experiments, and P values were determined using two-tailed Student’s t-test. Source numerical data are available in Source data.

Source data

Extended Data Fig. 4 Targeting G4 in specific genome loci by dCas9-mSA/Bio-PDC complexes.

a, The cytotoxicity of Bio-PDC in HEK293T, MCF-7, A375 and K562 cells was detected by CCK8 assays. b, Binding curves of Bio-PDC with a hairpin as the example of non-G4 structure, MycG4, KRAS G4, c-kit1 G4 and VEGFA G4 determined by ELISA. c,d, The FAM labelled G4s was captured by Bio-PDC through Magnetic streptavidin-coated beads (c), and the FAM signals were detected (d). e, The mRNA expression of MYC, KARS, KIT and VEGFA in HeLa cell treated with the indicated concentration Bio-PDC for 48 hours. Data are shown as mean ± SEM of three independent experiments, and P values were determined using two-tailed Student’s t-test. Source numerical data are available in Source data.

Source data

Extended Data Fig. 5 dCas9-mSA/Bio-PDC complexes guided by sgRNAs targeting MycG4 selectively inhibit MYC expression.

a, ChIP-qRT-PCR assays of dCas9-mSA constructs demonstrates correct localization to their intended genomic loci. b, The effects of Bio-PDC on EGFP expression in EGFP reporter expressed HEK293T cells. EGFP reporter gene was downstream of the MYC promoter with or without MycG4. c-e, The effects of Bio-PDC on EGFP expression in EGFP reporter expressed HEK293T cells transfecting with dCas9-mSA and sgR-3 (c), sg-4 (d) or sgR-6 (e). EGFP reporter gene was downstream of the MYC promoter with or without MycG4. f, The EGFP reporter expressed HEK293T cells pre-transfected with dCas9 or 4 MYC-targeting sgRNAs were treated with 500 nM Bio-PDC for 48 hours, and then the EGFP expression was detected. g, The occupancy of BG4 in the promoter region of MYC was measured by ChIP in HeLa cells transfected without or with dCas9-mSA and 4 MYC-targeting sgRNAs in response to 500 nM Bio-PDC for 48 hours. h, The mRNA expression of MYC, KARS, KIT, VEGFA and HRAS in HeLa cell transfected without or with dCas9-mSA and 4 MYC-targeting sgRNAs in response to 500 nM Bio-PDC for 48 hours. Data are shown as mean ± SEM of three independent experiments, and P values were determined using two-tailed Student’s t-test. Source numerical data are available in Source data.

Source data

Extended Data Fig. 6 Targeting G4 in specific genome loci by dCas9-SunTag-mSA/Bio-PDC complexes.

a, ChIP-qRT-PCR assays of dCas9-SunTag-mSA constructs demonstrates correct localization to their intended genomic loci. b, The mRNA expression of MYC in HeLa cells expressed with dCas9-mSA/4 sgRNAs or dCas9-SunTag-mSA/sgR-5 in response to 500 nM Bio-PDC for 48 hours. c, The occupancy of BG4 in the promoter region of MYC was measured by ChIP in HeLa cells expressed with dCas9-mSA/4 sgRNAs or dCas9-SunTag-mSA/sgR-5 in response to 500 nM Bio-PDC for 48 hours. d, The mRNA levels of VEGFA and HRAS in the HeLa cells expressed dCas9-SunTag-mSA/sgR-5 with the indicated treatment of PDC or Bio-PDC. e, The fold change of G4-driven oncogenes expression in the HeLa cells expressed dCas9-SunTag-mSA/sgR-5 in response to 500 nM Bio-PDC, 5 μM BRACO-19, 10 μM TMPyP4 and 10 μM DA3 (a G4-stabilized ligand with preference for MycG4) for 48 hours. Data are shown as mean ± SEM of three independent experiments, and P values were determined using two-tailed Student’s t-test. Source numerical data are available in Source data.

Source data

Extended Data Fig. 7 The formation of de novo G4s in vitro.

a, Genome browser tracks overlaid at GPX4, NFE2L2, VHL, MAPK1, NEAT1, MALAT1 genes. The orange box highlights the G4 sites, and the putative G4-forming sequences (PQSs) were shown. b, Circular dichroism spectroscopy of GPX4, NFE2L2, VHL, MAPK1, NEAT1, MALAT1 G4s and their mutants. c, Fluorescence turn-on assays of N-methyl mesophorphyrin IX (NMM) in the absence or presence of GPX4, NFE2L2, VHL, MAPK1, NEAT1, MALAT1 G4s and their mutants under indicated conditions. NMM, a well-known fluorescent G4-specific targeting compound. Source numerical data are available in Source data.

Source data

Extended Data Fig. 8 dCas9-SunTag-mSA guided Bio-PDC selectively stabilizes de novo G4.

a, Binding curves of Bio-PDC with GPX4, NFE2L2, VHL, MAPK1, NEAT1 and MALAT1 G4s determined by ELISA. b, The occupancy of BG4 in the promoter region of GPX4, NFE2L2, VHL, MAPK1, NEAT1 and MALAT1 was measured by ChIP in HeLa cells expressed with dCas9-SunTag-mS and sgRNAs targeting GPX4, NFE2L2, VHL, MAPK1, NEAT1 and MALAT1 G4s in response to 500 nM Bio-PDC for 48 hours. c, The Venn diagram shows the overlap of genes containing G4 motifs at the promoter region and differentially expressed genes. d, The Venn diagram shows the overlap of differentially expressed genes and the genes with differential ATAC signal in promoter. e, Genome browser tracks overlaid at KRAS and VEGFA genes. The brown box highlights the G4 sites. f, The occupancy of BG4 in the promoter region of PLEC and SEMA4B was measured by ChIP in HeLa cells expressed with dCas9-SunTag-mSA and two sgRNAs targeting NEAT1 and MALAT1 promoter G4s (sgR-lncRNAs) in response to 500 nM Bio-PDC for 48 hours. Data are shown as mean ± SEM of three independent experiments, and P values were determined using two-tailed Student’s t-test. Source numerical data are available in Source data.

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Extended Data Fig. 9 dCas9-SunTag-mSA/Bio-PDS complexes can stabilizes G4 in targeted genome loci.

a, Chemical structures of Bio-PDS. b,c The FAM labelled G4s was captured by Bio-PDS through Anti-Flag Magnetic Beads (b), and the FAM signals were detected (c). d, Inhibition of EGFP protein expression in the HEK293T cells expressed with dCas9-SunTag-mSA and sgR-5 by Bio-PDS concentration ranging from 50 to 1,000 nM treatment for 48 hours. e, The mRNA expression of MYC in HeLa cells expressed with dCas9-SunTag-mSA/sgR-5 in response to 200 nM Bio-PDS for 48 hours. f, The occupancy of BG4 in the promoter region of MYC was measured by ChIP in HeLa cells expressed with dCas9-SunTag-mSA/sgR-5 in response to 200 nM Bio-PDS for 48 hours. g, The mRNA levels of MYC and SRC in the HeLa cells expressed dCas9-SunTag-mSA/sgR-5 with the indicated treatment of PDS or Bio-PDS. h, Volcano plot (left) and heatmap (right) showing gene expression changes in HeLa cells transfected with dCas9-SunTag-mSA and sgR-lncRNAs in response to 200 nM Bio-PDS treatment for 48 hours. i, MA plots showing fold change of the expression of the genes with G4s in promoter. Data are shown as mean ± SEM of three independent experiments, and P values were determined using two-tailed Student’s t-test. Source numerical data are available in Source data.

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Extended Data Fig. 10 dCas9-SunTag-mSA/Bio-JQ1 complexes can activate gene transcription in targeted genome loci.

a, Chemical structures of Bio-JQ1. b, The mRNA expression of MyoD and CXCR4 in HEK293T cells transfected with dCas9-SunTag-mSA and sgR-MyoD or sgR-CXCR4 in response to 200 nM Bio-JQ1 treatment for 48 hours. Data are shown as mean ± SEM of three independent experiments, and P values were determined using two-tailed Student’s t-test. Source numerical data are available in Source data.

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Qin, G., Liu, Z., Yang, J. et al. Targeting specific DNA G-quadruplexes with CRISPR-guided G-quadruplex-binding proteins and ligands. Nat Cell Biol 26, 1212–1224 (2024). https://doi.org/10.1038/s41556-024-01448-1

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