CRISPR-assisted transcription activation by phase-separation proteins

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

Keywords

CRISPR / transcriptional activation / phase-separation proteins

References

[1] Adikusuma F, Piltz S, Corbett MA et al. Large deletions induced by Cas9 cleavage. Nature 2018;560:E8–E9.

[2] Ahn JH, Davis ES, Daugird TA et al. Phase separation drives aberrant chromatin looping and cancer development. Nature 2021;595:591–595.

[3] Altae-Tran H, Kannan S, Demircioglu FE et al. The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases. Science 2021;374:57–65.

[4] Baeumler TA, Ahmed AA, Fulga TA. Engineering synthetic signaling pathways with programmable dCas9-based chimeric receptors. Cell Rep 2017;20:2639–2653.

[5] Bai XT, Gu BW, Yin T et al. Trans-repressive effect of NUP98-PMX1 on PMX1-regulated c-FOS gene through recruitment of histone deacetylase 1 by FG repeats. Cancer Res 2006;66:4584–4590.

[6] Banani SF, Lee HO, Hyman AA et al. Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 2017;18:285–298.

[7] Becirovic E. Maybe you can turn me on: CRISPRa-based strategies for therapeutic applications. Cell Mol Life Sci 2022;79:130.

[8] Biamonti G, Vourc’h C. Nuclear stress bodies. Cold Spring Harb Perspect Biol 2010;2:a000695.

[9] Boija A, Klein IA, Sabari BR et al. Transcription factors activate genes through the phase-separation capacity of their activation domains. Cell 2018;175:1842–1855.e16.

[10] Boisvert FM, van Koningsbruggen S, Navascués J et al. The multifunctional nucleolus. Nat Rev Mol Cell Biol 2007;8:574–585.

[11] Borrow J, Shearman AM., Stanton VP Jr et al. The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9. Nat Genet 1996;12:159–167.

[12] Box JK, Paquet N, Adams MN et al. Nucleophosmin: from structure and function to disease development. BMC Mol Biol 2016;17:19.

[13] Brangwynne CP, Mitchison TJ, Hyman AA. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc Natl Acad Sci USA 2011;108:4334–4339.

[14] Brezgin S, Kostyusheva A, Kostyushev D et al. Dead Cas systems: types, principles, and applications. Int J Mol Sci 2019;20:6041.

[15] Capelson M, Liang Y, Schulte R et al. Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes. Cell 2010;140:372–383.

[16] Carter GC, Hsiung C-H, Simpson L et al. N-terminal domain of TDP43 enhances liquid-liquid phase separation of globular proteins. J Mol Biol 2021;433:166948.

[17] Chandra B, Michmerhuizen NL, Shirnekhi HK et al. Phase separation mediates NUP98 fusion oncoprotein leukemic transformation. Cancer Discov 2022;12:1152–1169.

[18] Charlesworth CT, Deshpande PS, Dever DP et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med 2019;25:249–254.

[19] Chavez A, Scheiman J, Vora S et al. Highly efficient Cas9-mediated transcriptional programming. Nature Methods 2015;12:326–328.

[20] Chen B, Gilbert LA, Cimini BA et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 2013;155:1479–1491.

[21] Chen S, Zhou Y, Chen Y et al. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018;34:i884–i890.

[22] Chen Y, Liu J, Zhi S et al. Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells. Nat Commun 2020;11:3136.

[23] Cho WK, Spille JH, Hecht M et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 2018;361:412–415.

[24] Chong S, Dugast-Darzacq C, Liu Z et al. Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 2018;361:eaar2555.

[25] Crozat A, Aman P, Mandahl N et al. Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma. Nature 1993;363:640–644.

[26] Cullot G, Boutin J, Toutain J et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nat Commun 2019;10:1136.

[27] Dominguez AA, Chavez MG, Urke A et al. CRISPR-mediated synergistic epigenetic and transcriptional control. Crispr J 2022;5: 264–275.

[28] Duronio RJ, Marzluff WF. Coordinating cell cycle-regulated histone gene expression through assembly and function of the Histone Locus Body. RNA Biol 2017;14:726–738.

[29] Falini B, Nicoletti I, Bolli N et al. Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias. Haematologica 2007;92:519–532.

[30] Fang X, Wang L, Ishikawa R et al. Arabidopsis FLL2 promotes liquid–liquid phase separation of polyadenylation complexes. Nature 2019;569:265–269.

[31] Feric M, Vaidya N, Harmon TS et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 2016;165:1686–1697.

[32] Gaudelli NM, Komor AC, Rees HA et al. Programmable base editing of A^*T to G^*C in genomic DNA without DNA cleavage. Nature 2017;551:464–471.

[33] Gilbert LA, Larson MH, Morsut L et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013;154:442–451.

[34] Gilbert LA, Horlbeck MA, Adamson B et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 2014;159:647–661.

[35] Gough SM, Slape CI, Aplan PD. NUP98 gene fusions and hematopoietic malignancies: common themes and new biologic insights. Blood 2011;118:6247–6257.

[36] Guo Y, Zhao S, Wang GG. Polycomb gene silencing mechanisms: PRC2 chromatin targeting, H3K27me3 ‘Readout’ and phase separation-based compaction. Trends Genet 2021;37: 547–565.

[37] Heman-Ackah SM, Bassett AR, Wood MJ. Precision modulation of neurodegenerative disease-related gene expression in human iPSC-derived neurons. Sci Rep 2016;6:28420.

[38] Hendel A, Bak RO, Clark JT et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 2015;33:985–989.

[39] Hille F, Richter H, Wong SP et al. The biology of CRISPR-Cas: backward and forward. Cell 2018;172:1239–1259.

[40] Hilton IB, D’Ippolito AM, Vockley CM et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 2015;33:510–517.

[41] Ibáñez de Opakua A, Geraets JA, Frieg B et al. Molecular interactions of FG nucleoporin repeats at high resolution. Nat Chem 2022;14:1278–1285.

[42] Jia W, Yao Z, Zhao J et al. New perspectives of physiological and pathological functions of nucleolin (NCL). Life Sci 2017;186:1–10.

[43] Kalverda B, Pickersgill H, Shloma VV et al. Nucleoporins directly stimulate expression of developmental and cell-cycle genes inside the nucleoplasm. Cell 2010;140:360–371.

[44] Kapitonov VV, Makarova KS, Koonin EV. ISC, a novel group of bacterial and archaeal DNA transposons that encode Cas9 homologs. J Bacteriol 2015;198:797–807.

[45] Kielkopf CL. Insights from structures of cancer-relevant pre-mRNA splicing factors. Curr Opin Genet Dev 2018;48:57–66.

[46] Kipniss NH, Dingal P, Abbott TR et al. Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system. Nat Commun 2017;8:2212.

[47] Knott GJ, Doudna JA. CRISPR-Cas guides the future of genetic engineering. Science 2018;361:866–869.

[48] Knott GJ, Bond CS, Fox AH. The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold. Nucleic Acids Res 2016;44:3989–4004.

[49] Koblan LW, Arbab M, Shen MW et al. Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning. Nat Biotechnol 2021;39:1414–1425.

[50] Komor AC, Kim YB, Packer MS et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 2016;533:420–424.

[51] Konermann S, Brigham MD, Trevino AE et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 2015;517:583–588.

[52] Koonin EV, Makarova KS, Zhang F. Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol 2017;37:67–78.

[53] Larson AG, Elnatan D, Keenen MM et al. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 2017;547:236–240.

[54] Liao HK, Hatanaka F, Araoka T et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 2017;171:1495–1507.e15.

[55] Lin DH, Hoelz A. The structure of the nuclear pore complex (an update). Annu Rev Biochem 2019;88:725–783.

[56] Lindsay ME, Plafker K, Smith AE et al. Npap60/Nup50 Is a Tri-stable switch that stimulates importin-α:β-mediated nuclear protein import. Cell 2002;110:349–360.

[57] Liu XS, Wu H, Ji X et al. Editing DNA methylation in the mammalian genome. Cell 2016;167:233–247.e17.

[58] Liu XS, Wu H, Krzisch M et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 Gene. Cell 2018;172:979–992.e6.

[59] Lyu XY, Deng Y, Huang XY et al. CRISPR FISHer enables high-sensitivity imaging of nonrepetitive DNA in living cells through phase separation-mediated signal amplification. Cell Res 2022;32:969–981.

[60] Machyna M, Neugebauer KM, Staněk D. Coilin: the first 25 years. RNA Biol 2015;12:590–596.

[61] Maeder ML, Linder SJ, Cascio VM et al. CRISPR RNA-guided activation of endogenous human genes. Nat Methods 2013;10:977–979.

[62] Makarova KS, Wolf YI, Iranzo J et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 2020;18:67–83.

[63] Makis AC, Chaliasos N, Hatzimichael EC et al. Recombinant human erythropoietin therapy in a transfusion-dependent beta-thalassemia major patient. Ann Hematol 2001;80:492–495.

[64] Mao D, Jia Y, Peng P et al. Enhanced efficiency of flySAM by optimization of sgRNA parameters in Drosophila. G3 Genes Genomes Genetics 2020;10:4483–4488.

[65] Martella A, Firth M, Taylor BJM et al. Systematic evaluation of CRISPRa and CRISPRi modalities enables development of a multiplexed, orthogonal gene activation and repression system. ACS Synth Biol 2019;8:1998–2006.

[66] Matharu N, Rattanasopha S, Tamura S et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science 2019;363:eaau0629.

[67] Michmerhuizen NL, Klco JM, Mullighan CG. Mechanistic insights and potential therapeutic approaches for NUP98-rearranged hematologic malignancies. Blood 2020;136:2275–2289.

[68] Mitrea DM, Cika JA, Guy CS et al. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. Elife 2016;5:e13571.

[69] Moore MS. Npap60: a new player in nuclear protein import. Trends Cell Biol 2003;13:61–64.

[70] Murakami T, Qamar S, Lin JQ et al. ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function. Neuron 2015;88:678–690.

[71] Nakamura T, Largaespada DA, Lee MP et al. Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nat Genet 1996;12:154–158.

[72] Niemelä E, Desai D, Lundsten E et al. Quantitative bioimage analytics enables measurement of targeted cellular stress response induced by celastrol-loaded nanoparticles. Cell Stress Chaperones 2019;24:735–748.

[73] Nizami Z, Deryusheva S, Gall JG. The Cajal body and histone locus body. Cold Spring Harb Perspect Biol 2010;2:a000653.

[74] Owen I, Yee D, Wyne H et al. The oncogenic transcription factor FUSCHOP can undergo nuclear liquid-liquid phase separation. J Cell Sci 2021;134:jcs258578.

[75] Pan C, Wu X, Markel K et al. CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants. Nat Plants 2021;7:942–953.

[76] Passon DM, Lee M, Rackham O et al. Structure of the heterodimer of human NONO and paraspeckle protein component 1 and analysis of its role in subnuclear body formation. Proc Natl Acad Sci USA 2012;109:4846–4850.

[77] Patel A, Lee HO, Jawerth L et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 2015;162:1066–1077.

[78] Patro R, Duggal G, Love MI et al. Salmon provides fast and biasaware quantification of transcript expression. Nat Methods 2017;14:417–419.

[79] Perez-Pinera P, Kocak DD, Vockley CM et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 2013;10:973–976.

[80] Qi LS, Larson MH, Gilbert LA et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013;152:1173–1183.

[81] Rabbitts TH, Forster A, Larson R et al. Fusion of the dominant negative transcription regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma. Nat Genet 1993;4:175–180.

[82] Raczynska KD, Ruepp MD, Brzek A et al. FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors. Nucleic Acids Res 2015;43:9711–9728.

[83] Riedmayr LM, Hinrichsmeyer KS, Karguth N et al. dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy. Nat Protoc 2022;17:781–818.

[84] Robinson MD, McCarthy DJ, Smyth GK. edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010;26:139–140.

[85] Ryan JJ, Sprunger ML, Holthaus K et al. Engineered protein disaggregases mitigate toxicity of aberrant prion-like fusion proteins underlying sarcoma. J Biol Chem 2019;294:11286–11296.

[86] Sabari BR, Dall’Agnese A, Boija A et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 2018;361:eaar3958.

[87] Savell KE, Bach SV, Zipperly ME et al. A neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation. eNeuro 2019;6.

[88] Schmidt HB, Görlich D. Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear porelike permselectivity. Elife 2015;4:e04251.

[89] Schuler G, Hu C, Ke A. Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9. Science 2022;376:1476–1481.

[90] Schwartz JC, Cech TR, Parker RR. Biochemical properties and biological functions of FET proteins. Annu Rev Biochem 2015;84:355–379.

[91] Shin Y, Berry J, Pannucci N et al. Spatiotemporal control of intracellular phase transitions using light-activated optodroplets. Cell 2017;168:159–171.e14.

[92] Singh RN, Howell MD, Ottesen EW et al. Diverse role of survival motor neuron protein. Biochim Biophys Acta Gene Regul Mech 2017;1860:299–315.

[93] Smith J, Calidas D, Schmidt H et al. Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3. Elife 2016;5:e21337.

[94] Soneson C, Love MI, Robinson MD. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res 2015;4:1521.

[95] Spannl S, Tereshchenko M, Mastromarco GJ et al. Biomolecular condensates in neurodegeneration and cancer. Traffic 2019;20:890–911.

[96] Takagi M, Sueishi M, Saiwaki T et al. A novel nucleolar protein, NIFK, interacts with the forkhead associated domain of Ki-67 antigen in mitosis. J Biol Chem 2001;276:25386–25391.

[97] Tanenbaum ME, Gilbert LA, Qi LS et al. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 2014;159:635–646.

[98] Tatomer DC, Terzo E, Curry KP et al. Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis. J Cell Biol 2016;213:557–570.

[99] Terlecki-Zaniewicz S, Humer T, Eder T et al. Biomolecular condensation of NUP98 fusion proteins drives leukemogenic gene expression. Nat Struct Mol Biol 2021;28:190–201.

[100] Trapnell C, Cacchiarelli D, Grimsby J et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol 2014;32:381–386.

[101] Tsai SQ, Joung JK. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev Genet 2016;17:300–312.

[102] Tsankov AM, Gu H, Akopian V et al. Transcription factor binding dynamics during human ES cell differentiation. Nature 2015;518:344–349.

[103] Uddin F, Rudin CM, Sen T. CRISPR gene therapy: applications, limitations, and implications for the future. Front Oncol 2020;10:1387.

[104] Wagner DL, Amini L, Wendering DJ et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat Med 2019;25:242–248.

[105] Wilsker D, Probst L, Wain HM et al. Nomenclature of the ARID family of DNA-binding proteins. Genomics 2005;86: 242–251.

[106] Wu, Z, Yang, H, Colosi, P. Effect of genome size on AAV vector packaging. Mol Ther 2010;18:80–86.

[107] Xu W, Pei G, Liu H et al. Compartmentalization-aided interaction screening reveals extensive high-order complexes within the SARS-CoV-2 proteome. Cell Rep 2021a;36:109482.

[108] Xu X, Chemparathy A, Zeng L et al. Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol Cell 2021b;81:4333–4345.e4.

[109] Yang L, Gal J, Chen J et al. Self-assembled FUS binds active chromatin and regulates gene transcription. Proc Natl Acad Sci USA 2014;111:17809–17814.

[110] Zhong S, Salomoni P, Pandolfi PP. The transcriptional role of PML and the nuclear body. Nat Cell Biol 2000;2:E85–E90.

[111] Zhou H, Liu J, Zhou C et al. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice. Nat Neurosci 2018;21:440–446.

[112] Zhou M, Li W, Li J et al. Phase-separated condensate-aided enrichment of biomolecular interactions for high-throughput drug screening in test tubes. J Biol Chem 2020;295:11420–11434.

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