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• W3135633881 abstract "•Cas9 can function as a programmable loader of superhelicase to genomic DNA•GOLD FISH locally unwinds target genomic DNA for FISH probe hybridization•GOLD FISH allows imaging of nonrepetitive genomic DNA with low background•GOLD FISH is scalable from targeting a 2.3-kb locus to chromosomal-scale “paint” Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue. Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue. The CRISPR-Cas9 system from Streptococcus pyogenes has been widely used for genome editing in cells (Cong et al., 2013Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9018) Google Scholar; Doudna and Charpentier, 2014Doudna J.A. Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9.Science. 2014; 346: 1258096Crossref PubMed Scopus (3118) Google Scholar; Mali et al., 2013bMali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (5898) Google Scholar). In the CRISPR-Cas9 system, the Cas9 endonuclease can be programed with a guide RNA to target a desired DNA sequence (Gasiunas et al., 2012Gasiunas G. Barrangou R. Horvath P. Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proc. Natl. Acad. Sci. USA. 2012; 109: E2579-E2586Crossref PubMed Scopus (1466) Google Scholar; Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (8083) Google Scholar; Sapranauskas et al., 2011Sapranauskas R. Gasiunas G. Fremaux C. Barrangou R. Horvath P. Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.Nucleic Acids Res. 2011; 39: 9275-9282Crossref PubMed Scopus (501) Google Scholar). An on-target DNA substrate of Cas9 ribonucleoprotein (RNP) contains a 20-nt protospacer region complementary to the spacer sequence of guide RNA and a protospacer-adjacent motif (PAM; 5′-NGG-3′ for S. pyogenes Cas9 [N representing any nucleotide]) (Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (8083) Google Scholar). After the Cas9 RNP binds to an on-target DNA substrate, the target strand (TS) and non-target strand (NTS) of the DNA substrate are cleaved by the HNH nuclease domain and RuvC nuclease domain, respectively (Gasiunas et al., 2012Gasiunas G. Barrangou R. Horvath P. Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proc. Natl. Acad. Sci. USA. 2012; 109: E2579-E2586Crossref PubMed Scopus (1466) Google Scholar; Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (8083) Google Scholar; Sapranauskas et al., 2011Sapranauskas R. Gasiunas G. Fremaux C. Barrangou R. Horvath P. Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.Nucleic Acids Res. 2011; 39: 9275-9282Crossref PubMed Scopus (501) Google Scholar). After catalysis in vitro, Cas9 remains stably bound to the cleaved DNA substrate (Singh et al., 2016Singh D. Sternberg S.H. Fei J. Doudna J.A. Ha T. Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9.Nat. Commun. 2016; 7: 12778Crossref PubMed Scopus (129) Google Scholar; Sternberg et al., 2014Sternberg S.H. Redding S. Jinek M. Greene E.C. Doudna J.A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.Nature. 2014; 507: 62-67Crossref PubMed Scopus (1041) Google Scholar). Engineering the active sites of the nuclease domains creates the Cas9dHNH variant (Cas9 with H840A mutation) that cuts only the NTS and the dCas9 variant (Cas9 with H840A and D10A mutations) that is inactive for DNA cleavage (Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (8083) Google Scholar). CRISPR-mediated transcriptional activation (CRISPRa) and inhibition (CRISPRi) platforms were developed utilizing dCas9 (Gilbert et al., 2013Gilbert L.A. Larson M.H. Morsut L. Liu Z. Brar G.A. Torres S.E. Stern-Ginossar N. Brandman O. Whitehead E.H. Doudna J.A. et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.Cell. 2013; 154: 442-451Abstract Full Text Full Text PDF PubMed Scopus (2018) Google Scholar; Maeder et al., 2013Maeder M.L. Linder S.J. Cascio V.M. Fu Y. Ho Q.H. Joung J.K. CRISPR RNA-guided activation of endogenous human genes.Nat. Methods. 2013; 10: 977-979Crossref PubMed Scopus (723) Google Scholar; Mali et al., 2013aMali P. Aach J. Stranges P.B. Esvelt K.M. Moosburner M. Kosuri S. Yang L. Church G.M. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nat. Biotechnol. 2013; 31: 833-838Crossref PubMed Scopus (1217) Google Scholar; Perez-Pinera et al., 2013Perez-Pinera P. Kocak D.D. Vockley C.M. Adler A.F. Kabadi A.M. Polstein L.R. Thakore P.I. Glass K.A. Ousterout D.G. Leong K.W. et al.RNA-guided gene activation by CRISPR-Cas9-based transcription factors.Nat. Methods. 2013; 10: 973-976Crossref PubMed Scopus (790) Google Scholar; Qi et al., 2013Qi L.S. Larson M.H. Gilbert L.A. Doudna J.A. Weissman J.S. Arkin A.P. Lim W.A. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.Cell. 2013; 152: 1173-1183Abstract Full Text Full Text PDF PubMed Scopus (2644) Google Scholar). DNA fluorescence in situ hybridization (FISH) allows for direct visualization of specific DNA sequences in situ, making it a powerful tool to study chromatin conformation and gene localization (Beliveau et al., 2012Beliveau B.J. Joyce E.F. Apostolopoulos N. Yilmaz F. Fonseka C.Y. McCole R.B. Chang Y. Li J.B. Senaratne T.N. Williams B.R. et al.Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes.Proc. Natl. Acad. Sci. USA. 2012; 109: 21301-21306Crossref PubMed Scopus (225) Google Scholar; Boettiger et al., 2016Boettiger A.N. Bintu B. Moffitt J.R. Wang S. Beliveau B.J. Fudenberg G. Imakaev M. Mirny L.A. Wu C.T. Zhuang X. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states.Nature. 2016; 529: 418-422Crossref PubMed Scopus (454) Google Scholar; Levsky and Singer, 2003Levsky J.M. Singer R.H. Fluorescence in situ hybridization: past, present and future.J. Cell Sci. 2003; 116: 2833-2838Crossref PubMed Scopus (361) Google Scholar; Wang et al., 2016Wang S. Su J.-H. Beliveau B.J. Bintu B. Moffitt J.R. Wu C.T. Zhuang X. Spatial organization of chromatin domains and compartments in single chromosomes.Science. 2016; 353: 598-602Crossref PubMed Scopus (281) Google Scholar). Conventional DNA FISH requires harsh conditions such as high temperature and concentrated formamide to globally denature genomic DNA for probe hybridization, which risk disrupting the integrity of biological structures such as heat-labile epitopes of proteins and increase the likelihood of DNA FISH probes binding to off-target genomic DNA sequences that are exposed due to global denaturing. By exploiting the high binding affinity of dCas9 RNP to specific DNA sequences, fluorescently labeled dCas9 RNP has been adopted for genomic loci imaging in live cells or in fixed cells without global genomic DNA denaturation (Chen et al., 2013Chen B. Gilbert L.A. Cimini B.A. Schnitzbauer J. Zhang W. Li G.W. Park J. Blackburn E.H. Weissman J.S. Qi L.S. Huang B. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.Cell. 2013; 155: 1479-1491Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar, Chen et al., 2016aChen B. Hu J. Almeida R. Liu H. Balakrishnan S. Covill-Cooke C. Lim W.A. Huang B. Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci.Nucleic Acids Res. 2016; 44: e75Crossref PubMed Scopus (111) Google Scholar; Deng et al., 2015Deng W. Shi X. Tjian R. Lionnet T. Singer R.H. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.Proc. Natl. Acad. Sci. USA. 2015; 112: 11870-11875Crossref PubMed Scopus (160) Google Scholar; Hong et al., 2018Hong Y. Lu G. Duan J. Liu W. Zhang Y. Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging.Genome Biol. 2018; 19: 39Crossref PubMed Scopus (33) Google Scholar; Ma et al., 2018Ma H. Tu L.C. Naseri A. Chung Y.C. Grunwald D. Zhang S. Pederson T. CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging.Nat. Methods. 2018; 15: 928-931Crossref PubMed Scopus (57) Google Scholar; Neguembor et al., 2018Neguembor M.V. Sebastian-Perez R. Aulicino F. Gomez-Garcia P.A. Cosma M.P. Lakadamyali M. (Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes.Nucleic Acids Res. 2018; 46: e30Crossref PubMed Scopus (20) Google Scholar; Qin et al., 2017Qin P. Parlak M. Kuscu C. Bandaria J. Mir M. Szlachta K. Singh R. Darzacq X. Yildiz A. Adli M. Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9.Nat. Commun. 2017; 8: 14725Crossref PubMed Scopus (126) Google Scholar; Wang et al., 2019Wang H. Nakamura M. Abbott T.R. Zhao D. Luo K. Yu C. Nguyen C.M. Lo A. Daley T.P. La Russa M. et al.CRISPR-mediated live imaging of genome editing and transcription.Science. 2019; 365: 1301-1305Crossref PubMed Scopus (95) Google Scholar). Visualization of nonrepetitive genomic sequences has been achieved with dCas9-binding-based genomic imaging methods (Chen et al., 2013Chen B. Gilbert L.A. Cimini B.A. Schnitzbauer J. Zhang W. Li G.W. Park J. Blackburn E.H. Weissman J.S. Qi L.S. Huang B. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.Cell. 2013; 155: 1479-1491Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar; Deng et al., 2015Deng W. Shi X. Tjian R. Lionnet T. Singer R.H. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.Proc. Natl. Acad. Sci. USA. 2015; 112: 11870-11875Crossref PubMed Scopus (160) Google Scholar; Hong et al., 2018Hong Y. Lu G. Duan J. Liu W. Zhang Y. Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging.Genome Biol. 2018; 19: 39Crossref PubMed Scopus (33) Google Scholar; Mao et al., 2019Mao S. Ying Y. Wu X. Krueger C.J. Chen A.K. CRISPR/dual-FRET molecular beacon for sensitive live-cell imaging of non-repetitive genomic loci.Nucleic Acids Res. 2019; 47: e131Crossref PubMed Scopus (14) Google Scholar; Qin et al., 2017Qin P. Parlak M. Kuscu C. Bandaria J. Mir M. Szlachta K. Singh R. Darzacq X. Yildiz A. Adli M. Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9.Nat. Commun. 2017; 8: 14725Crossref PubMed Scopus (126) Google Scholar; Shao et al., 2018Shao S. Chang L. Sun Y. Hou Y. Fan X. Sun Y. Multiplexed sgRNA Expression Allows Versatile Single Nonrepetitive DNA Labeling and Endogenous Gene Regulation.ACS Synth. Biol. 2018; 7: 176-186Crossref PubMed Scopus (17) Google Scholar), but the signal-to-background ratio was compromised by nonspecific binding of dCas9 RNP to off-targets in genomic DNA (Knight Spencer et al., 2017Knight Spencer C. Tjian R. Doudna Jennifer A. Genomes in Focus: Development and Applications of CRISPR-Cas9 Imaging Technologies.Angew. Chem. Int. 2017; 57: 4329-4337Crossref Scopus (40) Google Scholar; Lakadamyali and Cosma, 2020Lakadamyali M. Cosma M.P. Visualizing the genome in high resolution challenges our textbook understanding.Nat. Methods. 2020; 17: 371-379Crossref PubMed Scopus (31) Google Scholar; Wu et al., 2019Wu X. Mao S. Ying Y. Krueger C.J. Chen A.K. Progress and Challenges for Live-cell Imaging of Genomic Loci Using CRISPR-based Platforms.Genomics Proteomics Bioinformatics. 2019; 17: 119-128Crossref PubMed Scopus (38) Google Scholar). Cas9 RNP can tolerate up to 11 PAM-distal mismatches on the DNA substrate for stable binding in vitro (Singh et al., 2016Singh D. Sternberg S.H. Fei J. Doudna J.A. Ha T. Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9.Nat. Commun. 2016; 7: 12778Crossref PubMed Scopus (129) Google Scholar; Sternberg et al., 2014Sternberg S.H. Redding S. Jinek M. Greene E.C. Doudna J.A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.Nature. 2014; 507: 62-67Crossref PubMed Scopus (1041) Google Scholar). However, more than three PAM-distal mismatches drastically reduce or inhibit DNA cleavage activities of Cas9 RNP (Sternberg et al., 2015Sternberg S.H. LaFrance B. Kaplan M. Doudna J.A. Conformational control of DNA target cleavage by CRISPR-Cas9.Nature. 2015; 527: 110-113Crossref PubMed Scopus (311) Google Scholar), indicating that the cleavage specificity of Cas9 RNP is much higher than its specificity for stable binding. Conformational activation of Cas9 is dependent of the base-pairing between guide RNA and target DNA, but it is independent of whether the nuclease domains are engineered to be catalytically dead or not (Anders et al., 2014Anders C. Niewoehner O. Duerst A. Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.Nature. 2014; 513: 569-573Crossref PubMed Scopus (693) Google Scholar; Dagdas et al., 2017Dagdas Y.S. Chen J.S. Sternberg S.H. Doudna J.A. Yildiz A. A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9.Sci. Adv. 2017; 3: eaao0027Crossref PubMed Scopus (118) Google Scholar; Huai et al., 2017Huai C. Li G. Yao R. Zhang Y. Cao M. Kong L. Jia C. Yuan H. Chen H. Lu D. Huang Q. Structural insights into DNA cleavage activation of CRISPR-Cas9 system.Nat. Commun. 2017; 8: 1375Crossref PubMed Scopus (63) Google Scholar; Jiang et al., 2016Jiang F. Taylor D.W. Chen J.S. Kornfeld J.E. Zhou K. Thompson A.J. Nogales E. Doudna J.A. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.Science. 2016; 351: 867-871Crossref PubMed Scopus (301) Google Scholar; Nishimasu et al., 2014Nishimasu H. Ran F.A. Hsu P.D. Konermann S. Shehata S.I. Dohmae N. Ishitani R. Zhang F. Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA.Cell. 2014; 156: 935-949Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar; Sternberg et al., 2015Sternberg S.H. LaFrance B. Kaplan M. Doudna J.A. Conformational control of DNA target cleavage by CRISPR-Cas9.Nature. 2015; 527: 110-113Crossref PubMed Scopus (311) Google Scholar). We therefore expect that Cas9 nickase variants such as Cas9dHNH should have similar cleavage specificity as Cas9, and a genomic imaging method that relies on Cas9 or Cas9 nickase variants cleaving target DNA would have higher labeling specificity than Cas9-binding-based genomic imaging methods. In this study, we show that the post-cleavage Cas9dHNH-RNA-DNA ternary complex can recruit a 3′ to 5′ DNA helicase to unwind double-stranded DNA (dsDNA) beyond the protospacer. Exploiting this observation, we demonstrate a physiological-temperature DNA FISH method that leverages the high cleavage specificity of Cas9dHNH to label target genomic DNA. Previous cell-based studies suggested that the NTS in the Cas9-RNA-DNA complex is exposed and available for annealing to an exogenous DNA strand (Richardson et al., 2016Richardson C.D. Ray G.J. DeWitt M.A. Curie G.L. Corn J.E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA.Nat. Biotechnol. 2016; 34: 339-344Crossref PubMed Scopus (557) Google Scholar). Cleavage on the NTS reveals ∼17 nt of single-stranded DNA (ssDNA) with a 3′ hydroxyl end, the NTS 3′ flap (Figure 1A) (Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (8083) Google Scholar). A recent single-molecule study showed that the NTS 3′ flap can be digested by a ssDNA-specific exonuclease, suggesting the 3′ hydroxyl end may be exposed to solvent (Wang et al., 2021Wang Y. Mallon J. Wang H. Singh D. Hyun Jo M. Hua B. Bailey S. Ha T. Real-time observation of Cas9 postcatalytic domain motions.Proc. Natl. Acad. Sci. USA. 2021; 118 (e2010650118)Crossref Scopus (6) Google Scholar). Rep-X is a highly processive 3′ to 5′ DNA helicase engineered from E. coli Rep helicase through conformational control (Arslan et al., 2015Arslan S. Khafizov R. Thomas C.D. Chemla Y.R. Ha T. Protein structure. Engineering of a superhelicase through conformational control.Science. 2015; 348: 344-347Crossref PubMed Scopus (57) Google Scholar; Hua et al., 2018Hua B. Panja S. Wang Y. Woodson S.A. Ha T. Mimicking Co-Transcriptional RNA Folding Using a Superhelicase.J. Am. Chem. Soc. 2018; 140: 10067-10070Crossref PubMed Scopus (19) Google Scholar) based on mechanistic understanding of its activity regulation (Cheng et al., 2001Cheng W. Hsieh J. Brendza K.M. Lohman T.M. E. coli Rep oligomers are required to initiate DNA unwinding in vitro.J. Mol. Biol. 2001; 310: 327-350Crossref PubMed Scopus (117) Google Scholar; Comstock et al., 2015Comstock M.J. Whitley K.D. Jia H. Sokoloski J. Lohman T.M. Ha T. Chemla Y.R. Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme.Science. 2015; 348: 352-354Crossref PubMed Scopus (119) Google Scholar; Korolev et al., 1997Korolev S. Hsieh J. Gauss G.H. Lohman T.M. Waksman G. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP.Cell. 1997; 90: 635-647Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar; Lee et al., 2013Lee K.S. Balci H. Jia H. Lohman T.M. Ha T. Direct imaging of single UvrD helicase dynamics on long single-stranded DNA.Nat. Commun. 2013; 4: 1878Crossref PubMed Scopus (65) Google Scholar). The ssDNA translocating and dsDNA unwinding activities of Rep-X are powered by ATP hydrolysis. We hypothesized that Rep-X can be loaded onto the NTS 3′ flap, translocate along the NTS, and unwind the dsDNA downstream of the protospacer (Figure 1A). If this hypothesis is true, then Cas9 RNP can function as a programmable loader of Rep-X to genomic DNA, and the loaded Rep-X can unwind the downstream genomic DNA until it encounters an insurmountable blockade (Figure 1B). If Rep-X loaded onto a cleaved NTS unwinds a long enough stretch of genomic DNA, the resulting ssDNA could be targeted by fluorescently labeled oligonucleotide probes for site-specific imaging of genomic DNA in the cell (Figure 1B). In this scheme, which we call genome oligopaint via local denaturation (GOLD) FISH, after cell fixation, we first add the NTS nickase (Cas9dHNH RNP) to bind and cleave specific DNA sequences in the cells. Next, we add Rep-X and ATP to unwind the dsDNA downstream of each Cas9dHNH cleavage site and expose the FISH target strand (FISH-TS; Figure 1B). ATP is then removed. We surmised that the NTS that Rep-X translocates along may be removed from chromatin by Rep-X (e.g., if it hits another nick generated by Cas9dHNH nearby, forms secondary structures, or remains bound by Rep-X, preventing reannealing of the unwound genomic DNA) (Figure 1B). Finally, the Cy5-labeled FISH probes are added to hybridize with complementary FISH-TS. To test if Rep-X could be loaded onto the Cas9dHNH-generated NTS 3′ flap and unwind the dsDNA beyond the protospacer at the single-molecule level, we developed a DNA helicase invasion assay (Figure 1C). In this assay, the Cas9dHNH-RNA-DNA ternary complex was assembled in Mg2+-containing imaging buffer and immobilized on a quartz slide through biotin-NeutrAvidin interaction (Figure 1C). We internally labeled the PAM-distal region of the NTS with Cy5 so that if the loaded Rep-X fully unwound the 20-bp DNA downstream of the protospacer, then the fluorescence signal of Cy5 would be lost from the surface-tethered DNA (Figure 1C). The internal Cy5 labeling on the NTS does not affect the DNA cleavage activity of Cas9 (Singh et al., 2018Singh D. Wang Y. Mallon J. Yang O. Fei J. Poddar A. Ceylan D. Bailey S. Ha T. Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis.Nat. Struct. Mol. Biol. 2018; 25: 347-354Crossref PubMed Scopus (51) Google Scholar). When Rep-X and ATP were added together, the average number of fluorescent spots per image area decreased over time (Figures 1D and 1E), indicating that the Cy5-labeled DNA strand downstream of the NTS cleavage site was unwound by Rep-X and lost from the surface (Figure 1C). Control experiments using dCas9 or without ATP did not show Cy5 spots decrease with time (Figure 1F), indicating that both DNA nicking by Cas9dHNH and ATP hydrolysis-driven DNA unwinding by Rep-X are necessary to remove the downstream NTS DNA. Together, our data suggest that Rep-X can invade into the Cas9dHNH-RNA-DNA complex through the cleaved NTS and unwind the downstream dsDNA, supporting the design principle of GOLD FISH. We first tested GOLD FISH on a repetitive region within the MUC4 gene (MUC4-R) in IMR-90 cells, a human female diploid fibroblast strain (Nichols et al., 1977Nichols W.W. Murphy D.G. Cristofalo V.J. Toji L.H. Greene A.E. Dwight S.A. Characterization of a new human diploid cell strain, IMR-90.Science. 1977; 196: 60-63Crossref PubMed Scopus (281) Google Scholar), so that a single guide RNA and a single Cy5-labeled FISH probe could be used to decorate the gene with multiple fluorophores. The GOLD FISH images of MUC4-R obtained using epifluorescence microscopy showed that 93% of cells contained two to four bright foci with low background (Figures 2A and 2B ). The percentage of cells showing a particular number of foci is indicated above the corresponding histogram bin). We referred previously measured percentages of IMR-90 cells in G0/G1 (should have two foci) and S/G2/M (should have four foci) (Shi et al., 2007Shi X. Seluanov A. Gorbunova V. Cell divisions are required for L1 retrotransposition.Mol. Cell. Biol. 2007; 27: 1264-1270Crossref PubMed Scopus (69) Google Scholar), and estimated the efficiency of detection to be 90%. Control experiments performed using dCas9 or without ATP showed weak or no detectable foci (Figures S1A and S1B), indicating that both DNA nicking by Cas9dHNH and ATP hydrolysis-driven DNA unwinding by Rep-X are necessary to obtain bright GOLD FISH signals. To test if GOLD FISH works in other cell types and if the FISH probes co-localize with Cas9 binding sites, we used an ATTO550-labeled guide RNA and performed GOLD FISH of MUC4-R in HEK293ft cells (colocalization assay; Figure 2C). We found that the guide RNA, and by inference Cas9dHNH, remained visibly bound to target DNA under our experimental condition, and 90% of Cy5 loci co-localized with ATTO550 loci (Figures 2D and 2E). Next, we access whether GOLD FISH reduces nuclear background arising from nonspecific binding of Cas9dHNH RNP. Of note, it is possible that some on- and off-target ATTO550-labeled Cas9dHNH RNP dissociated from the cells during the Rep-X unwinding and FISH probe hybridization steps in GOLD FISH (Figure 2E). To fairly compare GOLD FISH signals with signals from labeled Cas9dHNH RNP binding, we also performed Cas9-mediated FISH (CASFISH) against MUC4-R using either Cas9dHNH or dCas9 with the ATTO550-labeled guide RNA (Figures S1C and S1D) (Deng et al., 2015Deng W. Shi X. Tjian R. Lionnet T. Singer R.H. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.Proc. Natl. Acad. Sci. USA. 2015; 112: 11870-11875Crossref PubMed Scopus (160) Google Scholar). We measured the signal-to-background ratios of the ATTO550 foci and Cy5 foci from the colocalization assay (S/BATTO550-colocalization and S/BCy5-colocalization), as well as that of the CASFISH foci (S/BCASFISH-Cas9dHNH and S/BCASFISH-dCas9) (Figure 2F). We found S/BCy5-colocalization (17.4 ± 6.6, mean ± SD) was substantially greater than S/BATTO550-colocalization (2.7 ± 1.4, mean ± SD), S/BCASFISH-Cas9dHNH (1.3 ± 0.8, mean ± SD), and S/BCASFISH-dCas9 (0.9 ± 0.4, mean ± SD). Control experiments performed without Cas9 enzyme showed that the nuclear background arising from cellular autofluorescence and nonspecific intracellular binding of ATTO550-labeled guide RNA was negligible (Figure S1E). Therefore, higher nonspecific binding of labeled Cas9dHNH RNP is responsible for the lower signal-to-background ratio of the ATTO550 foci in the colocalization assay and the CASFISH foci (Figure 2F). S/BATTO550-colocalization being higher than S/BCASFISH-Cas9dHNH also suggests that some nonspecifically bound Cas9dHNH RNP dissociated from the cells during the Rep-X unwinding and FISH probe hybridization steps in GOLD FISH (Figure 2F). Together, these data indicate that GOLD FISH, which requires Cas9 cleavage of target DNA to achieve efficient labeling (Figures 2B and S1A), has greatly reduced nonspecific labeling compared to genomic imaging methods that rely on labeled Cas9 RNP binding alone. Low nonspecific binding of GOLD FISH should greatly facilitate nonrepetitive loci imaging, which is generally much more challenging due to the need to include guide RNAs and FISH probes of multiple sequences at the same time. For example, if m different guide RNA sequences and n different FISH probes are used, then the total concentration of guide RNAs and FISH probes would have to be m and n times higher, respectively, to achieve the same signal level for each probe, potentially increasing background arising from nonspecific probe binding. A previous CASFISH study used 73 different guide RNAs to label a nonrepetitive region within the MUC4 gene and observed compromised labeling efficiency and increased background (Deng et al., 2015Deng W. Shi X. Tjian R. Lionnet T. Singer R.H. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.Proc. Natl. Acad. Sci. USA. 2015; 112: 11870-11875Crossref PubMed Scopus (160) Google Scholar). In order to test the capability of GOLD FISH in targeting nonrepetitive DNA sequences, we designed nine different guide RNAs (MUC4-NR guide RNA set 1) targeting a 2.3-kb nonrepetitive region within the MUC4 gene (MUC4-NR), with spacing of ∼300 bp between them, and 57 different Cy5-labeled FISH probes that bind regions between the guide RNAs (Figure 3A, top). Remarkably, GOLD FISH efficiently labeled the MUC4-NR region (Figure 3A); 89% of cells had two to four FISH loci, and the average signal-to-background ratio was 7.8 (Figures 3B and S2; the percentage of cells with a specific number of foci is shown above the histogram in Figure 3B). The specificity of MUC4-NR FISH loci was verified by colocalization with MUC4-R loci (Figures 3A and 3C). The excellent labeling efficiency and signal-to-background ratio of MUC4-NR GOLD FISH confirm that it is capable of nonrepetitive loci imaging without high nuclear background. Rep-X can unwind thousands of base pairs of dsDNA in vitro (Arslan et al., 2015Arslan S. Khafizov R. Thomas C.D. Chemla Y.R. Ha T. Protein structure. Engineering of a superhelicase through conformational control.Science. 2015; 348: 344-347Crossref PubMed Scopus (57) Google Scholar). To examine whether Rep-X is similarly processive on the genomic DNA, we performed GOLD FISH using the same fluorescently labeled probes targeting the MUC4-NR reg" @default.
• W3135633881 created "2021-03-15" @default.
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• W3135633881 date "2021-04-01" @default.
• W3135633881 modified "2023-10-15" @default.
• W3135633881 title "Genome oligopaint via local denaturation fluorescence in situ hybridization" @default.
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• W3135633881 doi "https://doi.org/10.1016/j.molcel.2021.02.011" @default.
• W3135633881 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8026568" @default.
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