G-quadruplex

Abstract

The invention relates to G-quadruplexes (G4s), and to novel compositions for binding to specific G4s in the genome. The invention also extends to the use of these compositions in methods of targeting specific G4s, as well as their use in methods of therapy and diagnosis.

Description

[0001] G-q ruplex The present invention relates to G-quadruplexes (G4s), and particularly, although not exclusively, to novel compositions for binding to specific G4s in the genome. The invention also extends to the use of these compositions in methods of targeting specific G4s, as well as their use in methods of therapy and diagnosis. Under physiological conditions, DNA can adopt non-canonical structures that deviate from the classical double-helix. Amongst these, G-quadruplexes (G4s) have recently emerged as interesting DNA secondary structures with the potential to regulate many different biological processes, including ageing and gene-expression. G-quadruplexes (G4s) can arise from guanine-rich sequences under physiological conditions (Figure 1). The formation of G4s in vitro has been widely characterised in the past few decades, but the actual formation of genomic G4s in the context of living cells has been long debated until recently. Immuno-precipitation experiments with a G4-selective antibody have revealed that G4-formation can be detected and mapped in human cells, suggesting their formation in living organisms. More recently, further evidence has been provided of G4-formation in living cells by developing a non- perturbative live-cell imaging method to visualise G4s, which has shown that the formation of these structures in human cells is highly dynamic, and enabled detection of ~3000 distinct G4s forming in a single cell. The simultaneous detection of thousands of G4s in a cell reflects the high prevalence of these structures in the human genome. Sequencing experiments have revealed that there are more than ~700,000 putative sequences in the human genome with the potential to form a G4. The high abundance of G4s makes G4-biology particularly challenging to characterise, as these structures are distributed in a variety of different genomic locations. For instance, enrichment of G4s has been observed at telomeric DNA, suggesting a potential role of these structures in regulating telomerase activity, which is relevant to cancer biology. Similarly, G4-forming sequences have been found to be highly enriched at gene promoters, which has the potential to regulate gene- expression and epigenetic pathways. Furthermore, recent studies from the inventors showed that specific G4s that form within ribosomal DNA act as natural substrates of CSB protein, which is involved in the development of the premature ageing disorder, Cockayne Syndrome. These findings suggest a potential role of ribosomal DNA G4s in ageing biology and highlights the high variability in biological relevance that these structures might have depending on their genomic location. Therefore, there is a clear need to tools for controlled targeting of specific G4s that can enable the study of individual G4-biology (e.g., telomeric vs ribosomal DNA G4s). Importantly, the lack of such tools prevents a detailed investigation and biological validation of G4-formation at different genomic locations, preventing their exploitation for therapeutic developments. So far, chemical approaches used to target G4s have generated a multitude of ligands with high selectivity for G4s versus double-stranded DNA, but no selectivity against the different types of G4s distributed across the genome. In fact, the design of selective G4- targeting agents is challenging and impractical, as it would require the development of ~700,000 distinct chemical structures. This highlights the need for a general platform that can be used to target any specific G4 of interest, enabling the unprecedented perturbation and characterisation of a single G4 at a time that can be leveraged for the discovery of new therapeutic targets. In view of the above, therefore, there is a need for novel strategies that can be explored to target specific G4s across the genome. Therefore, according to a first aspect of the invention, there is provided a CRISPR (clustered regularly interspaced short palindromic repeat) construct comprising a G- quadruplex (G4) binding ligand. Advantageously, the inventors have demonstrated that by conjugating a G4 binding ligand to a CRISPR construct, the G4 binding ligand can be positioned within the proximity of a particular G4 of interest using a dedicated, specifically designed short guiding RNA. As such, the inventors have developed a new platform that can be used to target any G4s within the human genome individually. The novel platform is called ATENA (Approach to Target Exact Nucleic Acid alternative structures), which relies on the chemical modification of established G4-binding ligands to enable their conjugation onto a catalytically inactive Cas9 protein (dCas9) using HaloTag, allowing for the targeting of individual G4s in living cells. This platform can then be used to characterise the diverse biological functions of different G4s and also identify novel G4s as potential therapeutic targets. CRISPR constructs may comprise at least an effector protein domain and / or either a guide RNA (gRNA) or a short guide RNA (sgRNA). The effector protein domain may comprise an enzyme that acts as a pair of molecular scissors capable of cutting a nucleic acid polymer (e.g., such as DNA or RNA) at a specific location so that pieces of nucleic acids can be added or removed, as desired. Target specificity may be controlled by the gRNA or sgRNA structu . The gRNA or sgRNA contains an approximately 20-nt target sequence at its 5’-end, which binds to a protospacer sequence in the target nucleic acid (e.g., DNA) that is adjacent to a protospacer adjacent motif (PAM). This guides the Cas protein (e.g., Cas9) to the protospacer sequences, adjacent the PAM sequence, for cleavage. Once at the target site, the Cas protein cuts the nucleic acid polymer (e.g., creates double strand breaks, DSB). The cell can be forced to repair the damaged nucleic acid via homology-directed repair by using a homologous nucleic acid donor template, which enables precise genomic modifications at the target site. Alternatively, repair will proceed via non-homologous end joining (NHEJ), which introduces random insertions or deletions (indels) of varying length at the site of the DSB. In some embodiments, the constructs described herein comprise one or more effector proteins. Any suitable effector protein may be used in the constructs described herein. In some embodiments, the effector protein may be any Class 2 CRISPR-Cas system, including any type II, type V, or type VI CRISPR-Cas enzyme. There have been continued developments in the nomenclature used to describe and / or identify CRISPR-Cas enzymes, such as Cas9 and Cas9 orthologs. For example, CRISPR-Cas nomenclature is discussed in Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems”: The CRISPR Journal, Vol.1. No.5, 2018. The particular CRISPR-Cas nomenclature used in any given instance herein will be understood by the skilled person. Without being bound by theory, the mechanism of action of certain effector proteins contemplated herein includes the step of forming an R-loop whereby the effector protein induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the effector protein. The gRNA or sgRNA spacer then hybridizes to the “target strand” at the 20-nt target sequence. This displaces a “non- target strand” that is complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the effector protein includes one or more nuclease activities, which then cut the DNA leaving various types of lesions. In some embodiments, the target DNA can be cut to form a “double-stranded break” whereby both strands are cut. In some embodiments, the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand. Exemplary effector proteins with different nuclease activities include “Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”). CRISPR-constructs may comprise the SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein—including any naturally occurring variant, mutant, or otherwise engineered version of Cas9. In some embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave one of the strands of the target DNA sequence. In some embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are “dead” Cas9 proteins. The CRISPR-constructs described herein may also comprise Cas9 equivalents, including Cas12a (Cpf1) and Cas12b1 proteins which are the result of convergent evolution. In some embodiments, the effector protein is a CRISPR-associated nuclease. As described herein, CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters may contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters may be transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA may require a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas (e.g., Cas9) protein. The tracrRNA may serve as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically may cleave linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA may be first cut endonucleolytically, then trimmed 3´-5′ exonucleolytically. In nature, DNA- binding and cleavage may require protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. In some embodiments, the effector protein directs cleavage of one or both strands at the location of a target sequence (e.g., near a G4 site), such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the effector protein directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes an effector protein that is mutated with respect to a corresponding wild-type enzyme such that the mutated effector protein lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A in reference to the canonical SpCas9 sequence, or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents. As used herein, the term “Cas protein” refers to a full-length Cas protein obtained from nature, a functionally active recombinant Cas protein having a sequences that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods, i.e., (i) possession of nucleic-acid programmable binding of the Cas protein to a target DNA, and (ii) ability to nick the target DNA sequence on one strand. The Cas proteins contemplated herein embrace CRISPR Cas 9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non- naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any Class 2 CRISPR system (e.g., type II, V, VI), including Cas12a (Cpf1), Cas12e (CasX), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), C2c4, C2c8, C2c5, C2c10, C2c9 Cas13a (C2c2), Cas13d, Cas13c (C2c7), Cas13b (C2c6), and Cas13b. Further Cas-equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the contents of which are incorporated herein by reference. The terms “Cas9” or “Cas9 nuclease” or “Cas9 moiety” or “Cas9 domain” embrace any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods. The term Cas9 is not meant to be particularly limiting and may be referred to as a “Cas9 or equivalent.” Exemplary Cas9 proteins are further described herein and / or are described in the art. The present disclosure is unlimited with regard to the particular Cas9 that is employed in the CRISPR-constructs of the invention. As noted herein, Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816- 821(2012), the entire contents of each of which are incorporated herein by reference). Examples of Cas9 and Cas9 equivalents are provided as follows; however, these specific examples are not meant to be limiting. The CRISPR-constructs of the present disclosure may use any suitable effector protein, including any suitable Cas9 or Cas9 equivalent. A. Wild type canonical SpCas9 In one embodiment, the primer editor constructs described herein may comprise the “canonical SpCas9” nuclease from S. pyogenes, which has been widely used as a tool for genome engineering and is categorized as the type II subgroup of enzymes of the Class 2 CRISPR-Cas systems. This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish one or both nuclease activities, resulting in a nickase Cas9 (nCas9) or dead Cas9 (dCas9), respectively, that still retains its ability to bind DNA in a sgRNA- programmed manner. When fused to another protein or domain, Cas9 or variant thereof (e.g., nCas9) may target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA. B. Wild type Cas9 orthologs In some embodiments, the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species different from the canonical Cas9 from S. pyogenes. For example, the following Cas9 orthologs can be used in connection with the CRISPR- constructs described in this specification: LfCas9 (Lactobacillus fermentum wild type), SaCas9 (Staphylococcus aureus wild type), SaCas9 (Staphylococcus aureus), StCas9 (Streptococcus thermophilus), LcCas9 (Lactobacillus crispatus), PdCas9 (Pedicoccus damnosus), FnCas9 (Fusobaterium nucleatum), EcCas9 (Enterococcus cecorum), AhCas9 (Anaerostipes hadrus), KvCas9 (Kandleria vitulina), EfCas9 (Enterococcus faecalis), Staphylococcus aureus Cas9,Geobacillus thermodenitrificans Cas9, or ScCas9 (Streptococcus Canis Cas9). In some embodiments, the CRISPR-constructs described herein include any of the ortholog sequences, or any variants thereof known to the skilled artisan. The effector protein may include any suitable homologs and / or orthologs or naturally occurring enzymes, such as, Cas9. Cas9 homologs and / or orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase. C. Dead Cas9 variant In certain embodiments, the CRISPR-constructs described herein may include a dead Cas9, e.g., dead SpCas9, which has no nuclease activity due to one or more mutations that inactive both nuclease domains of Cas9, namely the RuvC domain (which cleaves the non-20-nt target sequence DNA strand) and HNH domain (which cleaves the 20-nt target sequence DNA strand). The nuclease inactivation may be due to one or more mutations that result in one or more substitutions and / or deletions in the amino acid sequence of the encoded protein, or any variants thereof. As used herein, the term “dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9. Exemplary dCas9 proteins and method for making dCas9 proteins are further described herein and / or are described in the art. D. Cas9 nickase variant In one embodiment, the CRISPR-constructs described herein comprise a Cas9 nickase. The term “Cas9 nickase” of “nCas9” refers to a variant of Cas9 which is capable of introducing a single-strand break in a double strand DNA molecule target. In some embodiments, the Cas9 nickase comprises only a single functioning nuclease domain. The wild type Cas9 (e.g., the canonical SpCas9) may comprise two separate nuclease domains, namely, the RuvC domain (which cleaves the non-20-nt target sequence DNA strand) and HNH domain (which cleaves the 20-nt target sequence DNA strand). In one embodiment, the Cas9 nickase comprises a mutation in the RuvC domain which inactivates the RuvC nuclease activity. For example, mutations in aspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E) 762, have been reported as loss-of-function mutations of the RuvC nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et al., “Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935–949, which is incorporated herein by reference). Thus, nickase mutations in the RuvC domain may include D10X, H983X, D986X, or E762X, wherein X is any amino acid other than the wild type amino acid. In certain embodiments, the nicka s D10A, of H983A, or D986A, or E762A, or a combination thereof. In some embodiments, the CRISPR-constructs described herein include any Cas9 nickase sequence, or any variants thereof, known to the skilled artisan. E. Other Cas9 variants Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins used herein may also include other “Cas9 variants”, or fragments thereof, having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9. In some embodiments, the disclosure also may utilize Cas9 fragments which retain their functionality, and which are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. In some embodiments, the fragment is 100- 150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950- 1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, or 1250-1300 amino acids in length. In some embodiments, the CRISPR-constructs described herein include any Cas9 variant sequence known to the skilled artisan. F Cas9 equivalents In some embodiments, the CRISPR-constructs described herein can include any Cas9 equivalent. As used herein, the term “Cas9 equivalent” encompasses any effector protein that serves the same function as Cas9 in the present CRISPR-constructs even if its amino acid primary sequence and / or its three-dimensional structure may be different and / or unrelated from an standpoint. Thus, while Cas9 equivalents include any Cas9 ortholog, homolog, mutant, or variant described or embraced herein that are evolutionarily related, the Cas9 equivalents also embrace proteins that may have evolved through convergent evolution processes to have the same or similar function as Cas9, but which do not necessarily have any similarity with regard to amino acid sequence and / or three-dimensional structure. The CRISPR- constructs described here embrace any Cas9 equivalent that would provide the same or similar function as Cas9 despite that the Cas9 equivalent may be based on a protein that arose through convergent evolution. For instance, if Cas9 refers to a type II enzyme of the CRISPR-Cas system, a Cas9 equivalent can refer to a type V or type VI enzyme of the CRISPR-Cas system. For example, Cas12e (CasX) is a Cas9 equivalent that may have the same function as Cas9 but which evolved through convergent evolution. Thus, the Cas12e (CasX) protein described in Liu et al., “CasX enzymes comprises a distinct family of RNA- guided genome editors,” Nature, 2019, Vol.566: 218-223 , is contemplated to be used with the CRISPR-constructs described herein. In addition, any variant or modification of Cas12e (CasX) is conceivable and within the scope of the present disclosure. Cas9 is a bacterial enzyme that evolved in a wide variety of species. However, the Cas9 equivalents contemplated herein may also be obtained from archaea, which constitute a domain and kingdom of single-celled prokaryotic microbes different from bacteria. In some embodiments, Cas9 equivalents may refer to Cas12e (CasX) or Cas12d (CasY), which have been described in, for example, Burstein et al., “New CRISPR–Cas systems from uncultivated microbes.” Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR–Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR–Cas system. In bacteria, two previously unknown systems were discovered, CRISPR–Cas12e and CRISPR–Cas12d, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to Cas12e, or a variant of Cas12e. In some embodiments, Cas9 refers to a Cas12d, or a variant of Cas12d. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (effector protein), and are within the scope of this disclosure. Also see Liu et al., “CasX enzymes comprises a distinct family of RNA- guided genome editors,” Nature, 2019, . 66: 218-223. Any of these Cas9 equivalents are contemplated. In some embodiments, the Cas9 equivalent comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12e (CasX) or Cas12d (CasY) protein. In some embodiments, the effector protein is a naturally-occurring Cas12e (CasX) or Cas12d (CasY) protein. In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein. In some embodiments, the effector proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12e (CasX), Cas12d (CasY), Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a (C2c2), Cas12c (C2c3), Argonaute, and Cas12b1. One example of an effector protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (i.e, Cas12a (Cpf1)). Similar to Cas9, Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Cas12a (Cpf1) mediates robust DNA interference with features distinct from Cas9. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich 20-nt target sequence-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p.949-962. In some embodiments, the Cas protein may include any CRISPR associated protein, including but not limited to, Cas12a, Cas12b1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. In some embodiments, the effector be any of the following proteins: a Cas9, a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (CasY), a Cas12b1 (C2c1), a Cas13a (C2c2), a Cas12c (C2c3), a GeoCas9, a CjCas9, a Cas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, a Csn2, an xCas9, an SpCas9-NG, or an Argonaute (Ago) domain, or a variant thereof. Exemplary Cas9 equivalent protein sequences include the following: AsCas12a (Acidaminococcus sp. (strain BV3L6)), AsCas12a nickase (e.g., R1226A), LbCas12a (Lachnospiraceae bacterium GAM79), PcCas12a (Prevotella copri), ErCas12a (Eubacterium rectale), CsCas12a (Clostridium sp. AF34-10BH), BhCas12b (Bacillus hisashii), ThCas12b (Thermomonas hydrothermalis), LsCas12b (Laceyella sacchari), and DtCas12b (Dsulfonatronum thiodismutans) The CRISPR-constructs described herein may also comprise dead Cas12a (Cpf1) (dCpf1) variants that may be used as an effector protein. The Cas12a (Cpf1) protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cas12a (Cpf1) does not have the alfa-helical recognition lobe of Cas9. It was shown in Zetsche et al., Cell, 163, 759–771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cas12a (Cpf1) is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cas12a (Cpf1) nuclease activity. In some embodiments, the effector protein is a single effector of a microbial CRISPR- Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a (C2c2), and Cas12c (C2c3). Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cas12a (Cpf1) are Class 2 effectors. In addition to Cas9 and Cas12a (Cpf1), three distinct Class 2 CRISPR-Cas systems (Cas12b1, Cas13a, and Cas12c) have been described by Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov 5; 60(3): 385–397. Effectors of two of the systems, Cas12b1 and Cas12c, contain RuvC-like endonuclease domains related to Cas12a. A third system, Cas13a contains an effector with two predicated HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA- independent, unlike production of CRISPR RNA by Cas12b1. Cas12b1 depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial Cas13a has been shown to possess a unique RNase activity for CRI RNA maturation distinct from its RNA- activated single-stranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cas12a. See, e.g., East-Seletsky, et al., “Two distinct RNase activities of CRISPR-Cas13a enable guide-RNA processing and RNA detection”, Nature, 2016 Oct 13;538(7624):270-273, the entire contents of which are hereby incorporated by reference. In vitro biochemical analysis of Cas13a in Leptotrichia shahii has shown that Cas13a is guided by a single CRISPR RNA and can be programed to cleave ssRNA targets carrying complementary 20-nt target sequences. Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector”, Science, 2016 Aug 5; 353(6299), the entire contents of which are hereby incorporated by reference. The crystal structure of Alicyclobaccillus acidoterrastris Cas12b1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g., Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan 19;65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in Alicyclobacillus acidoterrestris C2c1 bound to target DNAs as ternary complexes. See e.g., Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec 15;167(7):1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2c1, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2c1-mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between C2c1 ternary complexes and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems. In some embodiments, the effector protein may be a C2c1, a C2c2, or a C2c3 protein. In some embodiments, the effector protein is a C2c1 protein. In some embodiments, the effector protein is a Cas13a protein. In some embodiments, the effector protein is a Cas12c protein. In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12b1 (C2c1), Cas13a (C2c2), or Cas12c (C2c3) protein. In some embodiments, the effector protein is a naturally-occurring Cas12b1 c1), Cas13a (C2c2), or Cas12c (C2c3) protein. In some embodiments, any one of the effector proteins disclosed herein may be fused to one or more other proteins to yield a fusion protein (e.g. the effector protein fused to the HALO tag). The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino- terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, an effector protein domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a reverse transcriptase. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). In some embodiments, any one of the effector proteins disclosed herein may be fused to any one of the G4 binding ligands, disclosed herein. In some embodiments, one or more G4 binding ligands may be fused to a single effector protein. The effector protein may comprise a CRISPR-associated endonuclease protein (i.e. a Cas protein or Cpf protein). CRISPR-associated endonuclease proteins are enzymes that bind and cleave a specific genomic site using a short RNA template (i.e. the guide RNA) that hybridises with a targeted DNA region of interest. The CRISPR-associated endonuclease protein may comprise a Cas protein. The Cas protein may be selected from a group consisting of: Cas1, Cas2, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas13, Csm, Cmr, and RNase III. In some embodiments, the Cas protein is a Cas9 protein. Alternatively, in another embodiment, the CRISPR-associated endonuclease protein may comprise a Cpf1 protein. In some embodiments, the present provides use of the inactive version of the endonuclease protein (e.g. deficient Cas9, i.e. dCas9), so that the function of the protein is limited to DNA binding only, thus acting as a carrier or scaffold for the G- quadruplex (G4) binding ligand and avoid damaging genomic DNA. Accordingly, in some embodiments, the effector protein comprises an endonuclease- deficient protein. In some embodiments, the effector protein may comprise endonuclease-deficient Cpf1. In some embodiments, the endonuclease-deficient protein comprises dCas9 protein. gRNA is an RNA sequence that recognises and is complementary to the target DNA region of interest. The guide RNA may comprise trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA). Alternatively, the guide RNA may comprise a single guide RNA (sgRNA). In some embodiments, the guide RNA comprises a sgRNA. The gRNA (or sgRNA) may recognise a target DNA region upstream or 5’ of a G- quadruplex. Alternatively, the gRNA (or sgRNA) may recognise a target DNA region downstream or 3’ of a G-quadruplex. In some embodiments, the gRNA (or sgRNA) recognises a target DNA region in close proximity to a G-quadruplex. In some embodiments, the gRNA (or sgRNA) recognises a target DNA region within at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides or at least 400 nucleotides of a G-quadruplex. In some embodiments, the gRNA (or sgRNA) recognises a target DNA region within at least 500 nucleotides, at least 1000 nucleotides, at least 1500 nucleotides or at least 2000 nucleotides of a G-quadruplex. In some embodiments, the gRNA (or sgRNA) recognises a target DNA region within at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides or at least 40 nucleotides of a G-quadruplex. In some embodiments, the gRNA (or sgRNA) recognises a target DNA region within at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, or at least 90 nucleotides of a G- quadruplex. In some embodiments, the gRNA (or sgRNA) recognises a target DNA region within at least at least 100 nucleotides of a G-quadruplex. G4s have been shown to be distributed in a variety of different genomic locations, such as within telomeric DNA, within gene promoters, within ribosomal DNA, or within a pseudogene. In some embodiments, the gRNA (or sgRNA) is not configured to recognise or target a DNA region that is coding. In some embodiments, the gRNA (or sgRNA) is not configured to recognise or target a DNA region that is an intron or exon in an ORF. In some embodiments, the gRNA (or sgRNA) is configured to recognise or target a DNA region that is non-coding. Advantageously, by avoiding coding sequences, gene function (i.e. encoded proteins) is not compromised. Accordingly, in some embodiments, the gRNA (or sgRNA) is configured to recognise or target a DNA region within telomeric DNA, a gene expression regulation region, a promoter region, ribosomal DNA, an enhancer, or a pseudogene. The gene expression regulation region may comprise a region of DNA to which any of the following regulator molecules may bind, i.e. a transcription factor, an activator, a repressor, an enhancer, a super-enhancer, a promoter, a specificity factor, an insulator, and / or a silencer. In some embodiments, the gRNA (or sgRNA) is configured to recognise or target a DNA region within a promoter. As described above, the gRNA contains a 20-nt target sequence known as a protospacer. The protospacer binds a DNA strand (in either the 3’-5’ or 5’-3’ direction) adjacent the PAM (e.g., the PAM immediately follows the targeted DNA sequence). The PAM is required for the nuclease to effect its function and is species specific. The most commonly used Cas9 nuclease recognises a PAM sequence of NGG that is found directly downstream (and adjacent) of the target sequence in the genomic DNA on the non-target strand. Recognition of the PAM by the nuclease is believed to destabilise the adjacent sequence, allowing interrogation of the sequence by the guide RNA, and resulting in RNA-DNA pairing when a matching sequence is present. The PAM is not present in the guide RNA sequence, but is in some embodiments immediately downstream of the target site in the genomic DNA. Therefore, the skilled person would understand that the guide RNA may further comprise a CRISPR nuclease binding sequence, e.g., a dCas9 nuclease binding sequence. The CRISPR nuclease binding sequence creates a secondary binding structure which complexes with the nuclease, for example a hairpin loop. The PAM on the host genome is recognised by the nuclease. In some embodiments, the G4 to which binding ligand can bind is located in a gene. In some embodiments, the G4 to which the G4 binding ligand can bind is located in a promoter of the gene. For example, the gene may be IL17RA. In some embodiments, the G4 to which the G4 binding ligand can bind is located in an oncogene. In some embodiments, the G4 to which the G4 binding ligand can bind is located in a promoter of the oncogene. For example, the oncogene may be the myc, N-MYC, PVT1, KRAS or c-KIT gene. In some embodiments, the G4 may be in the promoter of the myc, N-MYC, PVT1, KRAS or c-KIT gene. In some embodiments, the G4 site is on a coding strand (+, sense strand) of a DNA target molecule. In other embodiments, the G4 site is on an anti-coding strand (-). In some embodiments, the G4 sites are on both the coding and anti-coding strands. In some embodiments, the CRISPR construct is configured to target one or more G4s. For example, in one embodiment, the CRISPR construct is configured to target one or more G4s in a promoter region of a gene. In some embodiments, the CRISPR construct is configured to target one G4. Alternatively, the CRISPR construct is configured to target a plurality of G4s. For example, in one embodiment, the CRISPR construct is configured to target a plurality of G4s in a promoter region of a gene. It will be appreciated by the skilled person that a G-quadruplex (G4) binding ligand is a small molecule that can bind to a G-quadruplex. In some embodiments, the G4 binding ligand is non-specific, i.e. it will bind to any G4 within the genome. In some embodiments, the G4 binding ligand comprises pyridostatin (PDS), PhenDC3, CX5461, BRACO19, Pyrido Dicarboxamide (PDC), or Cl-PDC2, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. In some embodiments, the G4 binding ligand comprises pyridostatin (PDS), or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. In some embodiments, pyridostatin Formula (I): . In some embodiments, the G4 ligand comprises a pyridostatin-analogue comprising pyrrolidine sidechains (PyPDS), or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. In some embodiments, PyPDS comprises Formula (II): (II). In some embodiments, PhenDC3 comprises Formula (III): . Formula (IV):

[0002] In some embodiments, BRACO19 comprises Formula (V): . (PDC) comprises Formula (VIII): (VIII). In some embodiments, Cl-PDC2 comprises Formula (IX):

[0003] (IX). The term “pharmaceutically acceptable salt” may be understood to refer to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counter-ions well known in the art. Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, adepic, aspartic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2- ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4- methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic, 3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) base addition salts formed when an acidic proton present in the parent compound either (a) is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion or an aluminium ion, or alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminium, lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N′- dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)- aminomethane, tetramethylammonium hydroxide, and the like. Pharmaceutically acceptable salts may include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functionality, salts of n xic organic or inorganic acids, such as hydrohalides, e.g. hydrochloride, hydrobromide and hydroiodide, carbonate or bicarbonate, sulfate or bisulfate, borate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, sulfamate, nitrate, orotate, oxalate, palmitate, pamoate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, tannate, tartrate, tosylate, sorbate, ascorbate, malate, maleate, fumarate, tartarate, camsylate, citrate, cyclamate, benzoate, isethionate, esylate, formate, 3-(4- hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), methylsulphate, naphthylate, 2-napsylate, nicotinate, ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluceptate, gluconate, glucoronate, hexafluorophosphate, hibenzate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, muconate, xinofoate and the like. Hemisalts of acids and bases may also be formed, for example, hemisulphate salts. The skilled person will appreciate that the aforementioned salts include ones wherein the counterion is optically active, for example D-lactate, or racemic, for example DL- tartrate. The term “solvate” may be understood to refer to a compound provided herein or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate. Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D2O, d6-acetone and d6-DMSO. To ensure selective and cell-compatible conjugation of the G4 binding ligand onto the CRISPR construct, e.g., the effector protein thereof (e.g. a dCas9 protein), the inventors generated a conjugate or fusion of the effector protein (e.g. dCas9) with a moiety known as HALO-tag (thereby creating “dCas9-HALO”). It will be appreciated by the skilled person that HALO-tag is a known protein derived from haloalkane dehydrogenase enzyme DhaA from Rhodococcus rhodochrous. The active site of haloalkane dehydrogenase been genetically modified (Phe272 is substituted by His272), so that chloroalkane linker substrates can be irreversibly bound to the active site. Accordingly, HALO-tag is a protein that, under physiological conditions, reacts with a chloroalkane- (e.g., chlorohexane-) functionalised molecule, providing a strategy to create dCas9-(HALO)-G4 binding ligand conjugates with any G4 binding ligand containing a chloro-hexane moiety, as displayed in Figure 2A and 2B. In certain embodiments, the CRISPR construct according to the first aspect further comprises a modified haloalkane dehydrogenase moiety (i.e. a HALO-tag). In some embodiments, the modified haloalkane dehydrogenase moiety conjugates the G4 binding ligand to the CRISPR construct, e.g., the effector protein thereof, e.g., the dCas9 protein. In some embodiments, the modified haloalkane dehydrogenase moiety comprises at least one amino acid modification relative to a corresponding wildtype haloalkane dehydrogenase, wherein the modification of the modified haloalkane dehydrogenase allows a chloroalkane linker substrate to be irreversibly bound to its active site. In some embodiments, the modified haloalkane dehydrogenase moiety comprises an amino acid substitution at a position corresponding to amino acid residue 272 of Rhodococcus rhodochrous dehalogenase. In some embodiments, the substituted amino acid at the position corresponding to amino acid residue 272 is phenylalanine, glycine or alanine. The dCas9-HALO construct may comprise an amino acid sequence of SEQ ID No: 1, which is provided herein as follows: MKRPAATKKAGQAKKKKDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEEL LVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSE ETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGDSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGI AFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPN WLSTLEIS [SEQ ID No: 1] Accordingly, in one embodiment, the CRISPR construct comprises an amino acid sequence substantially as set out in SEQ ID No: 1, or a variant or fragment thereof. In order for the HALO-tag moiety to conjugate with the G4 binding ligand, the G4 binding ligand may be functionalised with a chloroalkane moiety. In some embodiments, the G4 binding ligand comprises or is functionalised with a chloroalkane moiety. In some embodiments, the G4 binding ligand may be functionalised with the chloroalkane moiety at any position of the G4 binding ligand. In some embodiments, the G4 binding ligand comprises or is functionalised with a PEG-chloroalkane moiety. The functionalised G4 binding ligand may be a compound of formula (VI): G4-L0-L1-L2-L3-L4-Cl (VI) or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein G4 is a G4 binding ligand; L0is absent or is an optionally substituted heteroatom; L1is absent or is an optionally substituted C1-20alkylene, an optionally substituted C2-20 alkenylene, an optionally substituted C2-20 alkynylene or a polyethylene glycol (PEG) group; L2is absent or is an optionally substituted heteroatom; L3is absent or is an optionally substituted C1-20 alkylene, an optionally substituted C2- 20 alkenylene, an optionally substituted C2-20 alkynylene or a polyethylene glycol (PEG) group; and L4is absent or is an optionally substituted C1-20alkylene, an optionally substituted C2-20 alkenylene or an optionally substituted C2-20 alkynylene. The inventors believe that they are the first to functionalise a G4 binding ligand with a chloroalkane moiety. Accordingly, in another aspect of the invention, there is provided a compound of formula (VI): G4-L0-L1-L2-L3-L4-Cl (VI) or pharmaceutically acceptable salt, sol , tautomeric form, stereoisomer or polymorphic form thereof, wherein G4 is a G4 binding ligand; L0is absent or is an optionally substituted heteroatom; L1is absent or is an optionally substituted C1-20 alkylene, an optionally substituted C2- 20 alkenylene, an optionally substituted C2-20 alkynylene or a polyethylene glycol (PEG) group; L2is absent or is an optionally substituted heteroatom; L3is absent or is an optionally substituted C1-20 alkylene, an optionally substituted C2- 20 alkenylene, an optionally substituted C2-20 alkynylene or a polyethylene glycol (PEG) group; and L4is absent or is an optionally substituted C1-20 alkylene, an optionally substituted C2- 20 alkenylene or an optionally substituted C2-20 alkynylene. An optionally substituted alkylene, alkenylene or alkynylene group may be unsubstituted or substituted with one or more OH or oxo groups. A heteroatom may be O, S or NH. In embodiments where the heteroatom is substituted, the heteroatom may be a nitrogen which is substituted. A heteroatom which is optionally substituted may unsubstituted or substituted with an optionally substituted C1-20 alkyl, an optionally substituted C2-20 alkenyl or an optionally substituted C2-20 alkynyl. The heteroatom which is optionally substituted may unsubstituted or substituted with an optionally substituted C1-10 alkyl, an optionally substituted C2-10 alkenyl or an optionally substituted C1-20 alkynyl. The heteroatom which is optionally substituted may unsubstituted or substituted with an optionally substituted C1-5 alkyl, an optionally substituted C2-5 alkenyl or an optionally substituted C1-5 alkynyl. An optionally substituted alkyl, alkenyl or alkynyl group may be unsubstituted or substituted with one or more OH or oxo groups.

[0004] G4 (i.e. the G4 binding ligand) may be , L0may be absent or may be O, S or NH. In some embodiments, L0is NH. In some embodiments, L0is absent. In some embodiments, L1is an optionally substituted C1-10 alkylene, an optionally substituted C2-10 alkenylene or an optionally substituted C2-10 alkynylene. In some embodiments, L1is an optionally substituted C3-7 alkylene, an optionally substituted C3-6 alkenylene or an optionally substituted C3-7 alkynylene. In some embodiments, the alkylene, alkenylene or alkynylene is independently substituted with one or two 1 L2may be absent or may be O, S or NH. In some embodiments, L2is NH. L3may be absent. Alternatively, L3may be a PEG group. Accordingly, L3may have , where n is an integer between 1 and 15, and the asterisk represents bonding to L4in embodiments where it is present, or to Cl in embodiments where L4is absent. In some embodiments, n is an integer between 1 and 10. In some embodiments, L4is an optionally substituted C1-10 alkylene, an optionally substituted C2-10 alkenylene or an optionally substituted C2-10 alkynylene. L4may be an optionally substituted straight chain alkylene, alkenylene or alkynylene. In some embodiments, L4is an optionally substituted alkylene. In some embodiments, L4is an optionally substituted C3-8 alkylene. In some embodiments, L4is –(CH2)6-. In some embodiments, the G4 binding ligand comprises pyridostatin (PDS), PhenDC3, CX5461, BRACO19, Pyrido Dicarboxamide (PDC), or Cl-PDC2, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. It will be appreciated by the skilled person that polyethylene glycol (PEG), is a hydrophilic polymer formed by a base-catalysed condensation reaction with repeating ethylene oxide units being added to ethylene. The molecular formula of PEG is (C2H4O)multH2O, where mult denotes the average number of oxyethylene groups. The inventors explored different PEG-linker lengths, in order to identify the ideal distance between the G4 binding ligand and the HALO-reactive moiety that ensures effective conjugation to the dCas9-HALO construct, without affecting the G4-binding properties of the ligands. In some embodiments, the polyethylene glycol (PEG) comprises one ethylene oxide unit, i.e. n may be 1 in the above formula. In some embodiments, the polyethylene glycol (PEG) comprises two or three ethylene oxide units, i.e. n may be 2 or 3. In some embodiments, the polyethylene glycol (PEG) comprises four or five ethylene oxide units, i.e. n may be 4 or 5. In som bodiments, the polyethylene glycol (PEG) comprises six or seven ethylene oxide units, i.e. n may be 6 or 7. In some embodiments, the polyethylene glycol (PEG) comprises eight ethylene oxide units, i,e, n may be 8. Alternatively, in some embodiments, the polyethylene glycol (PEG) comprises no ethylene oxide units, i.e. L3may be absent. In some embodiments, the polyethylene glycol (PEG) comprises four ethylene oxide units. The inventors have demonstrated the successful conjugation of a G4 binding ligand to a HALO-tag moiety, and believe that they are the first to conjugate these two compounds together. Accordingly, in a second aspect of the invention, there is provided a G-quadruplex (G4) binding ligand conjugated to a haloalkane dehydrogenase moiety. In some embodiments, the G4 binding ligand and the haloalkane dehydrogenase moiety are as described above for the first aspect. Accordingly, the conjugated compound may have formula (VII): G4-L0-L1-L2-L3-L4-HALO (VII) or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof wherein G4 and L0to L4are as defined above; and HALO is a haloalkane dehydrogenase moiety. It will be understood, therefore, that, in some embodiments, the G4 binding ligand comprises pyridostatin (PDS), PhenDC3, CX5461, BRACO19, Pyrido Dicarboxamide (PDC), or Cl-PDC2, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. In some embodiments, the G4 binding ligand comprises pyridostatin (PDS), or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. In some embodiments, pyridostatin or is a compound of Formula (I): . In some embodiments, the G4 binding ligand comprises a pyridostatin-analogue comprising pyrrolidine sidechains (PyPDS), or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. In some embodiments, PyPDS comprises or is a compound of Formula (II): (II). In some embodiments, the G4 binding ligand may be

[0005] . In some embodiments, PhenDC3 comprises or is a compound of Formula (III): . may be . In some embodiments, CX5461 compris is a compound of Formula (IV): . ligand may be In some embodiments, BRACO19 comprises or is a compound of Formula (V): . In some embodiments, the G4 binding may be . (PDC) comprises or is a compound of Formula (VIII): . In some embodiments, the G4 binding ligand may be Formula (IX): In some embodiments, the G4 binding ligand may be In some embodiments, the haloalkane dehydrogenase moiety comprises at least one amino acid modification relative to a corresponding wildtype haloalkane dehydrogenase, wherein the modification of the modified haloalkane dehydrogenase allows a chloroalkane linker substrate to be irreversibly bound to its active site. In some embodiments, the modified haloalkane dehydrogenase moiety comprises an amino acid substitution at a position corresponding to amino acid residue 272 of Rhodococcus rhodochrous dehalogenase. In some embodiments, the substituted amino acid at the position corresponding to amino acid residue 272 is phenylalanine, glycine or alanine. In some embodiments, the G4 binding ligand comprises or is functionalised with a chloroalkane moiety. In some embodiments, the G4 binding ligand comprises or is functionalised with a PEG-chloroalkane moiety. In some embodiments, the polyethylene glycol (PEG) comprises one ethylene oxide unit. In some embodiments, the polyethylene glycol (PEG) comprises two or three ethylene oxide units. In some embodiments, the polyethylene glycol (PEG) comprises four or five ethylene oxide units. In some embodiments, the polyethylene glycol (PEG) comprises six or seven ethylene oxide units. In some embodiments, the polyethylene glycol (PEG) comprises eight ethylene oxide units. Alternatively, in some embodiments, the polyethylene glycol (PEG) comprises no ethylene oxide units. In some embodiments, the polyethylene glycol (PEG) comprises four ethylene oxide units. The inventors have developed a highly innovative platform technology that can be used to target any specific G4 of interest within the genome. Accordingly, in a third aspect of the invention, there is provided a method of targeting a G-quadruplex (G4) in a DNA sequence, the method comprising contacting a DNA sequence comprising a target G4, with the CRISPR construct according to the first aspect. Advantageously, the inventors have demonstrated that by conjugating a G4 binding ligand to a CRISPR construct, the G4 binding ligand can be positioned within the proximity of a particular G4 of interest using a dedicated, specifically designed short guiding RNA. Accordingly, in some embodiments, the method comprises selectively targeting a G4 in a DNA sequence. In some embodiments, the target G4 is located in telomeric DNA, a gene expression regulation region, a promoter region, ribosomal DNA, an enhancer, or a pseudogene. In some embodiments, the target G4 is located in a gene or an oncogene. The gene may be IL17R. In some embodiments, the target G4 is located in a promoter of the oncogene. For example, the oncogene may be the myc, N-MYC, PVT1, KRAS or c-KIT gene. In some embodiments, the G4 may be in the promoter of the myc, N-MYC, PVT1, KRAS or c-KIT gene. Additionally, as demonstrated in the Examples, the inventors have shown that the CRISPR construct according to the invention is able to selectively target specific G4s (e.g. G4s in the promoter region of PVT-1), and as a result, perturb its endogenous expression levels. Accordingly, in a fourth aspect of the invention, there is provided a method of modifying gene expression, the method comprising contacting a gene comprising a target G-quadruplex (G4), with the CRISPR construct according to the first aspect. In some embodiments, the target G4 is located in a promoter region of the gene. The expression “modifying gene expression” may be an increase or a decrease in gene expression. The method according to the third or fourth aspect may be performed in vivo. In some embodiments, the method is performed in vitro or ex vivo. In some embodiments, the method is performed in vitro. Due to their potential to regulate many biological processes, G4s have been implicated in a number of diseases. Accordingly, due to its ability to target any specific G4 of interest anywhere in the genome of any cell, the CRISPR construct according to the invention may be used in the treatment of diseases mediated by a G4. Therefore, according to a fifth aspect of the invention, there is provided the CRISPR construct according to the first aspect, for use in therapy. Thus, in a sixth aspect of the invention, there is provided the CRISPR construct according to the first aspect, for use in treating, preventing or ameliorating a disease in a subject in need thereof. In some embodiments, the CRISPR construct according to the first aspect is for use in treating or ameliorating the disease in the subject in need thereof. In some embodiments, the CRISPR construct according to the first aspect is for use in preventing the disease in the subject in need thereof. In some embodiments, the disease is mediated by a G-quadruplex (G4). In a seventh aspect of the invention, there is provided a method of treating, preventing or ameliorating a disease in a subject in need thereof, the method comprising administering, or having administered, to the subject in need thereof an effective amount of the CRISPR construct according to the first aspect. In some embodiments, the method is a method of treating or ameliorating the disease in the subject in need thereof, the method comprising administering, or having administered, to the subject in need thereof a therapeutically effective amount of the CRISPR construct according to the first aspect. In some embodiments, the method is a method of preventing the disease in the subject in need thereof, the method comprising administering, or having administered, to the subject in need thereof a prophylactically effective amount of the CRISPR construct according to the first aspect. In some embodiments, the disease is mediated by a G-quadruplex (G4). It will be appreciated that the CRISPR construct according to the invention (referred to herein as “agent”) may be used in a monotherapy (e.g. the use of the CRISPR construct alone). Alternatively, the agent according to the invention may be used as an adjunct to, or in combination with, known therapies. The agent according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for exa , the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given. Medicaments comprising agents of the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents and medicaments of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin. Agents and medicaments according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection). In some embodiments, agents and medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion). It will be appreciated that the amount of the CRISPR construct (i.e. agent) that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the agent, and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease being treated. Additional factors depending on the particular subject being treated will result in a need to adjust including subject age, weight, gender, diet, and time of administration. Generally, a daily dose of between 0.001µg / kg of body weight and 10mg / kg of body weight of agent according to the invention may be used for therapy. In some embodiments, the daily dose of agent is between 0.01µg / kg of body weight and 1mg / kg of body weight, e.g., between 0.1µg / kg and 100µg / kg body weight, e.g., between approximately 0.1µg / kg and 10µg / kg body weight. The agent may be administered before, during or after onset of the disease. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the agent may require administration twice or more times during a day. As an example, agents may be administered as two (or more depending upon the severity of the disease being treated) daily doses of between 0.07 µg and 700 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, the agent may require administration once a week for even once a month. Alternatively, a slow release device may be used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the agents according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration). In an eighth aspect of the invention, there is provided a pharmaceutical composition comprising the CRISPR construct according to the first aspect, and optionally a pharmaceutically acceptable vehicle. The invention also provides in a ninth aspect, a process for making the pharmaceutical composition according to the eighth aspect, the process comprising combining a therapeutically effective amount of the CRISPR construct according to the first aspect, with a pharmaceutically acceptable vehicle. A “subject” may be a vertebrate, mammal, or domestic animal. Hence, medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. In some embodiments, the subject is a human. A “therapeutically effective amount” of the CRISPR construct is any amount which, when administered to a subject, is the amount that is needed to treat the disease being treated, or produce the desired effect. A “prophylactically effective amount” of the CRISPR construct is any amount which, when administered to a subject, is the amount that is needed to prevent the disease being prevented, or produce the desired effect. For example, the therapeutically effective amount of the CRISPR construct used may be from about 0.001 ng to about 1 mg, e.g., from about 0.01 ng to about 100 ng. In some embodiments, the amount of the CRISPR construct is an amount from about 0.1 ng to about 10 ng, e.g., from about 0.5 ng to about 5 ng. A “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions. In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets may contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like. However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehic ch as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, e.g., sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. The agents and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The agents used according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions. Additionally, by detecting specific G4s in the genome, it may be possible to diagnose or prognose a condition in a subject. Thus, advantageously, the CRISPR construct of the invention may also be used as a diagnostic tool. Accordingly, in a tenth aspect, there is the CRISPR construct according to the first aspect, for use in diagnosing or prognosing a disease in a subject. In some embodiments, the CRISPR construct according to the first aspect, is for use in diagnosing or prognosing a disease in a subject. In some embodiments, the disease is mediated by a G-quadruplex (G4). In an eleventh aspect, there is provided a method of diagnosing or prognosing a disease in a subject, the method comprising detecting a G quadruplex (G4) in a biological sample obtained from the subject with the CRISPR construct according to the first aspect. Prognosis may relate to determining the therapeutic outcome in a subject that has been diagnosed with a disease. Prognosis may relate to predicting the rate of progression or improvement and / or the duration of disease in a subject, the probability of survival, and / or the efficacy of various treatment regimes. Thus, a poor prognosis may be indicative of disease progression, low probability of survival and reduced efficacy of a treatment regime. A favourable prognosis may be indicative of disease resolution, high probability of survival and increased efficacy of a treatment regime. The diagnostic method may be performed in vivo. In some embodiments, the method of diagnosis is performed in-vitro or ex-vivo. In some embodiments, the method of diagnosis is performed in-vitro. Additionally, by detecting specific G4s in the genome, it may be possible to identify and / or validate new therapeutic targets. Accordingly, in a twelfth aspect of the invention, there is provided a method of identifying and / or validating a G-quadruplex (G4) as a therapeutic target, the method comprising contacting a DNA sequence comprising a target G4, with the CRISPR construct according to the first aspect, the G-quadruplex (G4) binding ligand according to the second aspect, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof as described herein. In another aspect, there is provided a method of identifying and / or validating a G- quadruplex (G4) as a therapeutic target based on the expression levels of a gene of interest, the method comprising contacting a gene of interest comprising a target G4, with the CRISPR construct according to rst aspect, the G-quadruplex (G4) binding ligand according to the second aspect, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof as described herein. As such, it is possible to identify therapeutic relevance based on the cellular and transcriptional context of the G4. The biological sample may be any material that is obtainable from a subject. The biological sample may be tissue or a biological fluid. Furthermore, the sample may be blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, breast milk, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, lymph, granuloma, cerebrospinal fluid, or combinations thereof. The biological sample may comprise blood, urine, tissue etc. In some embodiments, the biological sample comprises a blood sample. The blood may be venous or arterial blood. Blood samples may be assayed immediately. Alternatively, the blood sample may be stored at low temperatures, for example in a fridge or even frozen before the method is conducted. Alternatively, the blood sample may be stored at room temperature, for example between 18 to 22 degrees Celsius, before the method is conducted. The blood sample may comprise blood serum. The blood sample may comprise blood plasma. Alternatively, the detection is carried out on whole blood or the blood sample is peripheral blood. The blood may be further processed before the diagnostic method is performed. For instance, an anticoagulant, such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added. It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms “substantially the amino acid / nucleotide / peptide sequence”, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequences of of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No: 1, and so on. Amino acid / polynucleotide / polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein. The skilled technician will appreciate how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. In order to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants. Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment. Preferably, calculation of percentage identities between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N / T)*100. Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence, which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45ºC followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65ºC. Alternatively, a substantially similar peptide may differ by at least 1, 2, 3, 4 or 5 amino acids from the sequences shown in SEQ ID No: 1. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids. All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:- Figure 1 illustrates a structural (top) and a schematic (bottom) representation of a DNA G-quadruplex (G4). Figure 2 shows a schematic representation of one embodiment of a CRISPR construct according to the invention. A) shows a dCas9 protein that enables positioning of the G4-ligand within the proximity of a G4 of interest using a dedicated short guiding RNA (sgRNA). B) shows the conjugation of the G4-ligand onto the dCas9 protein using a HALO-tag, to create a dCas9-(HALO)-G4-ligand. Figure 3 shows A) the chemical structure of a PyPDS G4-binding scaffold (top). Amide coupling functionalisation of PyPDS containing a HALO-reactive side-chain (bottom). B) Mechanism of HALO functionalization of the HALO with the functionalized G4- ligands described. Figure 4 illustrates a general strategy for PyPDS synthesis. PyPDS was synthesised through the amide coupling of the central pyridine 4 and two-mole equivalents of quinoline 1, followed by the Boc-deprotection. Prior to the amide coupling, the quino- line and pyridine were each decorated with different side chains and synthesised, respectively. Figure 5 illustrates quinoline scaffold The quinoline 1 was synthesised by the functionalisation of 2-amino-quinolinone with 1-(2-hydroxyethyl)-pyrrolidine through a Mitsunobu reaction using triphenylphosphine and diisopropyl azodicarboxylate (DIAD). Figure 6 shows pyridine scaffold (4) synthesis. The two carboxylic acids of chelidamic acid were protected with methanol in the presence of concentrated sulfuric acid to give the ester 2. Then the Mitsunobu reaction was performed on the phenolic alcohol of 2 with N-Boc-ethanolamine to afford compound 3. The subsequent deprotection of 3 with hydroxide ions afforded the central dicar-boxylic acid 4. Figure 7 shows synthesis of PEG-chlorohexane linkers with PEG linker lengths (n=2 and 4). PEG4-chlorohexane 11 (n=4) was synthesised starting from the commercially available Boc-protected poly-ethylene glycol. Nucleophilic substitution was performed in the presence of the strong base NaH to remove the alcoholic proton and attach the chlorohexane onto the PEG chain. Following the Boc-deprotection by TFA, the resulting amine attacks glutaric anhydride catalysed by DMAP to produce a terminal carboxylic acid, which can participate in the subsequent amide coupling. PEG2-chlorohexane 7 (n=2) was synthesised from the amino intermediate 2-(2-((6-chlorohexyl)oxy)ethoxy) ethan-1-amine and the same procedures were followed. Figure 8 shows the synthesis of PEG0-chlorohexane linker. PEG0-chlorohexane was synthesised following a modified scheme starting with 6-amino-1-hexanol. Chlorination on the primary alcohol in the starting material was done using thionyl chloride to obtain 13. Then following the same reaction with glutaric anhydride, carboxylic acid 14 was generated. Figure 9 shows synthesis of PyPDS-PEG-chlorohexane (n=0, 2, 4) through amide coupling using HATU. Figure 10 shows the results from protein purification. (A) 8% SDS-PAGE (Coomassie stained) analysis of His-Tag IMAC purified dCas9Halo. After cell homogenisation, cell debris was assessed on SDS-PAGE, and the supernatant was purified by immobilised metal affinity chromatography (IMAC). Wash fractions (W1-3) and elution fractions (E1-E4) were indicated at the top of the gel. Bands at expected sizes (182 kDa) were indicated by the arrow. (B) Elution profile of dCas9Halo eluted from IEC column with 0.1-1M KCl gradient. (C), (D), (E) and (F) 8% SDS-PAGE (Coomassie stained) analysis of samples without IEC purification, unb sample (FT) and relevant fractions from IEC. (G) Combined fractions B1-D7 after IEC purification. Figure 11 shows the specific labelling of HaloTag on dCas9Halo in vitro. (A) Chemical structure of tetramethylrhodamine (TAMRA). (B) SDS-PAGE analysis (Coomassie stained and in-gel fluorescence) of three batches of dCas9Halo after labelling by the TAMRA ligand for an hour at room temperature. Batch I: 5 μΜ of protein with both IMAC and IEC purification. Batch II: 1 μΜ of protein with IMAC purification only. Batch III: 10 μM of protein with IMAC purification only. Figure 12 shows a schematic illustration of sgRNA design for targeting G- quadruplexes within the PVT-1 promoter using one embodiment of the CRISPR construct according to the invention. Figure 13 illustrates (A) the G4 binding ligand PyPDS-PEG0-chlorohexane and (B) gene expression changes of PVT-1, ELL and LRP1 genes, after 2 hours of treatment with the CRISPR construct according to the invention and a further 6 hours of incubation. Figure 14 illustrates (A) the G4 binding ligand PyPDS-PEG2-chlorohexane and (B) gene expression changes of PVT-1, ELL and LRP1 genes, after 2 hours of treatment with the CRISPR construct according to the invention, and a further 6 hours of incubation. Figure 15 illustrates (A) the G4 binding ligand PyPDS-PEG2-chlorohexane and (B) gene expression changes of PVT-1, ELL and LRP1 genes, after 2 hours of treatment with the CRISPR construct according to the invention, and a further 18 hours of incubation. Figure 16 illustrates (A) the G4 binding ligand PyPDS-PEG4-chlorohexane and (B) gene expression changes of PVT-1, ELL and LRP1 genes, after 2 hours of treatment with the CRISPR construct according to the invention, and a further 6 hours of incubation. Figure 17 illustrates (A) the G4 binding ligand PyPDS-PEG4-chlorohexane and (B) gene expression changes of PVT-1, ELL and LRP1 genes, after 2 hours of treatment with the CRISPR construct according to the invention, and a further 18 hours of incubation. Figure 18 illustrates chemical labeling of dCas9 for selective G4-targeting. a, Schematic representation of a G-Tetrad via Hoogsteen base pairing with central cation (K+) (modified from PDB file:6W9P) (Left) and G-Tetrad stacking to form G4-structure (modified from PDB file:6W9P using Protein Imager33). (Right). b, Schematic overview of ATENA (Approach to Target Exact Nucleic Acid alternative structures): dCas9-Halo fusion protein functionalized with chloroalkane-modified G4-ligands enables single G4-targeting through sgRNA guidance. c, Chemical structure of chloroalkane-modified PyPDS where n indicates the different PEG linkers length (Cl- PDSn). d, Chemical structure of chloroalkane-modified PhenDC3 (Cl-PhenDC3n) where n indicates the different PEG linkers length (Cl-PhenDC3n). e, FRET-melting assay of 4 µM Cl-PDS2 or 4 µM Cl-PDS4 with 0.4 µM of dually labeled BCL-2 G4-forming sequence (left) or hTelo –G4 forming sequence (right). ^^Tm indicates the difference in melting temperature between folded and unfolded G4. The mean is calculated from two independent experiments (n=2). f, Illustration of the competition assay workflow to test the binding ability of Cl-PDSn probes to dCas9-Halo purified recombinant protein. g, SDS-Page gel of the Cl-PDSn competition assay shows each sample’s fluorescent level acquired in the TAMRA channel (542 nm) and the corresponding protein level (Coomassie staining). h, Schematic representation of the dually labeled FRET oligos containing c-kit–G4 forming sequence bound by dCas9-Halo labeled with Cl-PDSn probes to study G4 stabilization with respect to the PEG-linker length and sgRNAs. i, ∆FRET efficiency. The values indicated were extrapolated from the band intensity (ImageStudio Software) measured in the Cy3 channel (Typhoon FLA 9500- 550 nm). Specifically, the DFRET efficiency was calculated as follows ((1-(Cy3 band intensity in presence of Cl-PDSn / Cy3 band intensity samples without molecule)) and normalized for the non-targeting sgRNA control (n=2). Figure 19 illustrates ATENA enables selective targeting of a G4 in the c-MYC promoter. a, Schematic representation of CAPA assay (left). CAPA assay results were obtained upon treatment of MCF7 cells stably expressing dCas9-Halo to assess Cl- PDSn label efficiency in cells at different concentrations (n=2). Flow cytometry was performed using Attune NxT Flow cytometer, and analysis was performed using FlowJo software (right). b, Schematic illustration of the c-MYC promoter with annotated G4, sgRNA targeting region (black triangles), and their relative distance in bp from the G4. c, RT-qPCR for c-MYC expression in MCF7 cells stably expressing dCas9-Halo transfected with the indicated sgRNAs and incubated for 48h in the presence of 2.5 µM of Cl-PDS2. The expression values are represented as fold change (2-ΔΔCt) with respect to the non-treated transfected samples and after normalization for a housekeeping gene (GAPDH). n=3, biological of which included two technical replicates. d, RT-qPCR for c-MYC expression in MCF7 cells stably expressing dCas9- Halo transfected with sgRNAMYC+58 and incubated for 24h in the presence of either Cl- PDS2 (2.5 µM) or Cl-Oregon green (5 µM). The expression values are represented as fold change (2-ΔΔCt) with respect to the non-treated transfected samples and after normalization for a housekeeping gene (GAPDH). n=2, biological replicates, each of which includes two technical replicates. e, RT-qPCR for P1-driven c-MYC expression in MCF7 cells stably expressing dCas9-Halo transfected with the indicated sgRNAs and incubated for 48h in the presence of 2.5 µM of Cl-PDS2. The expression values are represented as fold change (2-ΔΔCt) with respect to the non-treated but transfected samples and after normalization for a housekeeping gene (GAPDH). n=3, biological replicates, each of which includes two technical replicates. f, BG4 CUT&Tag-qPCR for MCF7 cells stably expressing dCas9-Halo transfected with either sgRNAMYC-19 or sgRNA NTC and treated with DMSO or 2.5 µM Cl-PDS2. BG4 accessibility was analyzed for c- MYC and normalized to three G4s in control gene sites (RPA3, MAZ, RBBP4). n=2, biological replicates for each sample three technical replicates for BG4 and one for the negative (no BG4 treatment). g, RT-qPCR for P1 dependent c-MYC expression in MCF7 cells stably expressing dCas9-Halo transfected with sgRNAMYC-19 or sgRNA NTC and incubated for 48h in the presence of 2.5 µM of Cl-PDS2. The expression values are represented as fold change (2-ΔΔCt) with respect to the non-treated but transfected samples and after normalization for a housekeeping gene (GAPDH). n=3, biological replicates, each of which includes two technical replicates. Data presented are mean of n = number of independent biological samples. Statistical significance was calculated using a two-tailed t-test. Only significant values are represented P-value: ns > 0.05, * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. Fold change (FC) was calculated on the normalized expression value. Figure 20 illustrates ATENA unveils ligand-dependent transcriptional response of the lncPVT1. a, Schematic overview of the PVT1 promoter containing annotation of the predicted G4, sgRNA targeting region (black triangles) and their relative distance in bp from the G4 forming sequence b, RT-qPCR for PVT1 expression in MCF7 cells stably expressing dCas9-Halo, transfected with either sgRNAPVT1-20 ,sgRNAPVT1+33 or sgRNA NTC and treated with 2.5 µM of Cl-PDS2 for 48h after transfection. The expression values are represented as fold change (2-ΔΔCt) with respect to the non-treated transfected samples and after normalization for a housekeeping gene (GAPDH). n=3, biological replicates, each of which included two technical replicates. c, BG4 CUT&Tag- qPCR for MCF7 cells stably expressing dCas9-Halo transfected with either sgRNAPVT1-20 or sgRNA NTC and treated with DMSO or 2.5 µM Cl-PDS2. BG4 accessibility was analyzed for PVT1 and normalized to th 4s in control gene sites (RPA3, MAZ, RBBP4). n=2, biological replicates, including three technical replicates for BG4 and one for the negative (no BG4 treatment). d, RT-qPCR for PVT1 expression in MCF7 cells stably expressing dCas9-Halo, transfected with either sgRNAPVT1-20 , sgRNAPVT1+33 or sgRNA NTC and treated with 2.5 µM of Cl-PhenDC32 for 48h after transfection. Values are represented as fold change (2-ΔΔCt) with respect to the DMSO samples and after normalization for a housekeeping gene (GAPDH). n=3, biological replicates, each of which includes two technical replicates. e, (Top) Chemical structure of chloroalkane- modified PDC (Cl-PDC2) with two PEG linkers length. e, (bottom) RT-qPCR for PVT1 expression in MCF7 cells stably expressing dCas9-Halo, transfected with either sgRNAPVT1-20 or sgRNA NTC and treated with 2.5 µM of Cl-PDC2 for 48h after transfection. Values are represented as fold change (2-ΔΔCt) with respect to the DMSO samples and after normalization for a housekeeping gene (GAPDH). n=3, biological replicates each of which includes two technical replicates. Data presented are mean of n = number of independent biological samples. Statistical significance was calculated using a two-tailed t-test. P-value: ns > 0.05, * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. Fold change (FC) was calculated on the normalized expression values. Figure 21 illustrates targeting of de novo G4s with ATENA uncovers a transcriptional- dependent functional response. A schematic overview of the HMGN1 promoter contains annotation of the predicted G4, two sgRNA designed to target HMGN1 G4 (complementary) (black triangles), and their relative distance in bp from the G4 forming sequence. b, RT-qPCR for HMGN1 expression in MCF7 cells stably expressing dCas9-Halo, transfected with either sgRNAHMGN1-22, sgRNAHMGN1+34 or sgRNA NTC and treated with 2.5 µM of Cl-PDS2 for 48h after transfection. Values are represented as fold change (2-ΔΔCt). The expression values are represented as fold change (2-ΔΔCt) with respect to the non-treated transfected samples and after normalization for a housekeeping gene (GAPDH). n=2, biological replicates, each of which includes two technical replicates. c, RT-qPCR for IL17RA expression in MCF7 cells stably expressing dCas9-Halo, transfected with either sgRNAIL17RA-20 or sgRNA NTC and treated with 2.5 µM of Cl-PDS2 for 48h after transfection. Values are represented as fold change (2-ΔΔCt). The expression values are represented as fold change (2-ΔΔCt) with respect to the non-treated transfected samples and after normalization for a housekeeping gene (GAPDH) n=2, biological replicates, each of which includes two technical replicates. Data presented are mean of n = number of independent biological samples. Statistical significance was calculated using a two-tailed t-test. P-value: ns > 0.05, * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. Fold change (FC) was calculated on the normalized expression value. Examples The inventors hypothesised that different G4s can regulate diverse biological processes based on their genomic location, and set out to develop a platform technology that can characterise such diverse biological functions and identify novel G4s as potential therapeutic targets. To achieve this selectivity, the inventors conjugated known G4-binding ligands (i.e. Pyridostatin analogues) to an endonuclease-deficient dCas9 protein that enables the positioning of the G4-ligand within the proximity of a particular G4 of interest using a dedicated short guiding RNA (Figure 2A). This is based on the powerful genome-editing technology, CRISPR-Cas9. Cas9 is a nuclease enzyme that normally binds and cleaves a specific genomic site using a short RNA template that hybridises with the targeted DNA region of interest. However, by using the inactive (endonuclease deficient) version of Cas9 (dCas9), the inventors limited the function of the protein to DNA binding only, thus acting only as a carrier / scaffold for the G4-binding ligand. Chemical conjugation of the dCas9 protein with a G4-binding ligand therefore carries the G4-interacting molecule onto a specific G4 of interest by the use of rationally designed RNA templates that localise the dCas9- G4-ligand conjugate within proximity of the specific G4-structure of interest (Figure 2). To ensure selective and cell-compatible incorporation of the G4-ligand onto the dCas9 protein, the inventors have generated a genetic fusion of dCas9 with HALO-tag (dCas9-HALO, Figure 2). HALO-tag is a commercial protein that, under physiological conditions, reacts with chloro-hexane-functionalised molecules, providing a practical strategy to create dCas9-(HALO)-G4-ligand conjugates with any G4-ligand containing a chloro-hexane moiety, as displayed in Figure 2A. The dCas9-G4-ligand conjugate can then be used to target the G4 of interest by rational design of appropriate short- guiding RNAs to position the dCas9 complex in proximity of the selected G4 (Figure 2). This innovative approach enables the interrogation of the biological functions of individual G4s in living cells for the first time (Figure 2A), which can be used to identify new therapeutic targets. Materials and Methods To generate one embodiment of the dCas9-G4 selective platform according to the invention, the inventors developed a strategy based on three key objectives: 1. Synthesis of a small library of G4-binding ligands (G4 ligands), which were exemplified using pyridostatin analogues. These G4 ligands were decorated with HALO-reactive side-chains t ere used to functionalise the dCas9-HALO protein. 2. Expression and purification of the dCas9-HALO protein to screen and validate the ability of the G4 ligands to generate a stable covalent complex with the protein in vitro. 3. Cellular validation of the developed dCas9-G4-ligand platform for selective targeting of the well-characterised G4-structure present in the promoter region of the PVT-1 gene, using stable cell lines that constitutively express dCas9- HALO. This served as a proof-of-concept to demonstrate selective engagement with the targeted G4s over the other G4s present elsewhere in the human genome. Example 1 - Synthesis of G4-ligands reactive towards HALO-tag The inventors first set out to generate new G4-ligands functionalised with a chloro- hexane moiety to allow fusion with a HALO-tagged dCas9 protein, as schematically depicted in Figure 2. The inventors developed novel pyridostatin (PDS) decorated with a HALO-tag reactive moiety (chloro-hexane), using the amide-coupling strategies displayed in Figure 3. Briefly, the synthetic strategy is based on the synthesis of a PDS-analogue containing pyrrolidine side-chains (PyPDS, Figure 3), which can be functionalised by reacting the primary amine in the scaffold by amide coupling (Figure 3), without perturbing the G4-binding ability of the ligand. Therefore, the invention described herein leverages this chemistry to reliably obtain PDS-analogues functionalised with a HALO-reactive moiety (Figure 3). Importantly, the inventors have explored several different lengths of PEG-linkers (Figure 3), which allows identification of the ideal distance between the G4-binding ligand and the HALO-reactive moiety that ensures effective conjugation to the dCas9- HALO construct, without affecting the G4-binding properties of the ligands (Figure 3). Moreover, different linkers can be used to optimize G4-targeting depending on the specific genomic location of interest in which the G4 site is located. Chemical Synthesis Three analogues of PyPDS-PEG-chlorohexane were produced with PEG linker lengths of 0, 2, and 4. The analogues were synthesised by the amide coupling of the amine (PyPDS) and the carboxylic acid (PEG-chlorohexane). PyPDS was first synthesised in a six-step synthetic scheme adapted from the literature with minor changes. Then the PEG-chlorohexane chain was synthesised by attaching the chlorohexane to repeating PEG units through nucleophilic by the ring opening of glutaric anhydride to afford a terminal carboxylic acid for the subsequent amide coupling. PyPDS PyPDS was synthesised through the amide coupling of the central pyridine 4 and two- mole equivalents of quinoline 1, followed by the Boc-deprotection (Figure 4). Prior to the amide coupling, the quinoline and pyridine were each decorated with different side chains and synthesised, respectively. Quinoline Scaffold The quinoline 1 was synthesised by the functionalisation of 2-amino-quinolinone with 1-(2-hydroxyethyl)-pyrrolidine through a Mitsunobu reaction using triphenylphosphine and diisopropyl azodicarboxylate (DIAD) (Figure 5). While the chemical transformation went as expected, the direct purification was unsuccessful following column chromatography and required further optimisation. Impurities remained after column chromatography were removed by co-evaporation. The 1H NMR analysis on the elutes from column chromatography revealed that the major impurity was unreacted pyrrolidine. Although the boiling point of pyrrolidine was low, strong intermolecular interactions between pyrrolidine and the desired product caused chromatography purification to be unsuccessful. Co-evaporation was done by dissolving the crude in DCM and placing it under the high vacuum after the solvent evaporated at room temperature. The process was repeated three to four times to afford the pure quinoline 1 with a product yield of 54%. Pyridine Scaffold Pyridine 4 was prepared from the chelidamic acid (Figure 6). The serial reaction started with the protection on the two carboxylic acids of chelidamic acid with methanol in the presence of concentrated sulfuric acid to give the ester 2. Then the Mitsunobu reaction was performed on the phenolic alcohol of 2 with N-Boc- ethanolamine to afford compound 3 with a product yield of 58%. The subsequent deprotection of 3 with hydroxide ions afforded the central dicar-boxylic acid 4 with a 63% yield. The first reaction, which was the protection of carboxylic acid as dimethyl ester from subsequent Mitsunobu reaction, was amended to avoid using a large volume of SOCl2(100 mL) and was replaced with a catalytic amount of sulfuric acid. It gave a more than two-fold increase in the percent yield (from 29% to 64%) as well as a safer working condition. The Mitsunobu reacti , ven though effective, produces by- products with similar chromatography mobility that necessitate large efforts for purification using column chromatography. In the interest of time, the purification protocol was not optimised for this study. However, it is worth trying to remove one of the by-products triphenylphosphine oxide by precipitation with CaBr2 to afford an easier column for future purification. PyPDS coupling To obtain PyPDS, acyl chloride was generated in situ by treating acid pyridine 4 with Ghosez’s reagent, and then coupled to the amine quinoline 1 in the presence of triethylamine to form the Boc-protected PyPDS (Figure 4). The isolation of the product required precipitation with acetonitrile, which resulted in significant difficulties. The precipitation did not work with a reaction scale larger than 100 mg of the starting reagent 4, in which case one or more impurities would precipitate together with product 5 as indicated in TLC. With a scale of 100 mg or lower, precipitation with acetonitrile afforded pure product 5 with the highest yield of 32%. In fact, the yield was rather low with 10-20% most of the time, and experimenting with different conditions had improved the yield slightly. It was found that a concentrated reaction system with minimum DCM could facilitate the reaction because the double carboxylic acids have a higher barrier to be activated and coupled simultaneously, and the mono- substituted intermediate was formed in the reaction indicated by LC / MS. LC / MS therefore can be used to monitor the formation of activated acid 4 at both sites before the addition of triethylamine and amine 1 to ensure sufficient equivalents of coupling reagent and reaction time. Finally, NHBoc-PyPDS 5 was deprotected with trifluoroacetic acid (TFA) to obtain PyPDS salt with a quantitative yield, which was taken forward to the coupling with the HaloTag reactive linker. PyPDS-PEG-Chlorohexane PEG-chlorohexane PEG4-chlorohexane 11 (n=4) was synthesised starting from the commercially available Boc-protected poly-ethylene glycol (Figure 7). Nucleophilic substitution was performed in the presence of the strong base NaH to remove the alcoholic proton and attach the chlorohexane onto the PEG chain. Following the Boc-deprotection by TFA, the resulting amine can then attack glutaric anhydride catalysed by DMAP to produce a terminal carboxylic acid, which can participate in the subsequent amide coupling. PEG2-chlorohexane 7 (n=2) was from the amino intermediate 2-(2-((6- chlorohexyl)oxy)ethoxy) ethan-1-amine and the same procedures were followed (Figure 7). Synthesis of PEG-chlorohexane linkers where PEG linker lengths are 2 and 4 (n=2 and 4) PEG0-chlorohexane was synthesised following a modified scheme starting with 6- amino-1-hexanol (Figure 8). Chlorination on the primary alcohol in the starting material was done using thionyl chloride to obtain 13. Then following the same reaction with glutaric anhydride, carboxylic acid 14 was generated. While all reactions were successful, the yields were limited due to the complicated purification processes. Preparative LC / MS was used to purify 7 and resulted in a low yield of 14.6%. However, an alternative extraction method was discovered during the purification of PEG4 acid 11. A basic extraction with NaHCO3 deprotonated the carboxylic acid and dragged it into the aqueous layer while the majority of impurities remained in the organic layer. Then the product was recovered into the organic layer after acidifying with 10% HCl. Although the product obtained was impure as observed in the analytical LC / MS, the purity was acceptable, and more of the compound was obtained to carry onto the next reaction. The same was performed for PEG0 acid 14 to obtain a satisfying purity indicated by analytical LC / MS. PyPDS-PEG-chlorohexane coupling The final probe PyPDS-PEG-chlorohexane (n=0, 2, 4) was synthesised through the amide couplings of PyPDS and PEG-chlorohexane linker using the coupling reagent HATU (Figure 9). HATU has a high coupling efficiency and fast reaction rate. During the reaction, an activated OAt-ester is generated and is believed to form a stable transition state with the neighbouring pyridine group, conferring a high reactivity. LC / MS was used to monitor the reaction progress and C18 reverse-phase column chromatography was used to purify the compounds 8, 12, and 15. Compounds with a shorter PEG linker (n=0) gave a better separation in the column chromatography, while those with longer PEG linkers (n=2, 4) might require more than one run of column chromatography to acquire a complete separation. Example 2 – Biophysical validation and optimisation of dCas9-G4-ligand conjugates dCas9-Halo Recombinant dCas9Halo protein was using a bacterial expression system. After obtaining copies of the DNA encoding for the protein of interest, the plasmid was transformed into bacterial cells. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added when the optical density of the culture reached ~0.6, to activate the lac promoter on the plasmid and initiate gene expression and protein production. Protein of interest was harvested by cell lysis, homogenisation, and centrifugation. An initial purification was performed using cobalt IMAC resin. The successful protein expression and purification quality of the fractions collected from IMAC were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 10). His6-tagged proteins, including the protein of interest dCas9Halo, have a high affinity for cobalt immobilised on resin, so cobalt IMAC resin was used to isolate the protein of interest from all other proteins present in bacteria. Proteins unbound to cobalt resin, lacking a His6-tag, were collected in flow through. Proteins that weakly bound to cobalt non-specifically were interfered and washed off in fractions W1-W3, using low concentration of imidazole. By increasing the concentration of imidazole to 150 mM, strongly bound His-tagged dCas9Halo was eluted in fractions E1-E4. Ion exchange chromatography (IEC) was applied to further purify the proteins. As imidazole introduced during His-Tag purification affects the equilibration of the IEC column and the overall performance of the purification, it was removed before this step by buffer exchange. In addition, fractions E1-E4 from His-Tag purification were combined and loaded onto the negatively charged column. As the theoretical pI of dCas9Halo is 4.56, the buffer used was at pH 7.5 to ensure successful binding of positively charged dCas9Halo to negatively charged resin. After binding of dCas9-Halo and the resin due to electrostatic interactions, a continuous gradient concentration of KCl between 0.1M to 1M was applied to dCas9Halo to elute it by competition. dCas9Halo was recovered in a single peak (Figure 10B), suggesting that most dCas9Halo was eluted by the time when the gradient of KCl had reached 50%. However, the tailing peak indicated that there were still proteins that are not fully recovered. The reason could be that the KCl in the elution buffer was not concentrated enough to wash off all dCas9Halo. The quality of the purification of all fractions under the main peak were verified using SDS-PAGE (Figure 10C-F). The presence of dCas9Halo in the eluted fractions was confirmed. The band at expected size in the flow through revealed the presence of unbound proteins (Figure 11B). Figure 11F shows an analysis of combination of fractions B1-D7 from IEC. The 2-step purification by IMAC and IEC did improve the purity of dCas9Halo, compared to proteins only purified by IMAC (Figures 10C and 10G), very effective in eliminating proteins smaller than 50 kDa. The inventors assessed the purity after IEC as acceptable for downstream experiments, although an extra step of purification (e.g. size exclusion chromatography) would provide an optimal level of protein purity. dCas9Halo is prone to precipitation. Therefore, for final concentration of proteins using ultra centrifugal filters, small amounts of protein at a time are better to process to prevent precipitation. With both the dCas9-HALO fusion and the G4-ligands library developed, the inventors’ second key milestone was to investigate the G4-binding properties of the generated ligands when conjugated to the dCas9-HALO protein and guided towards a G4- structure of interest using an appropriate sgRNA. To achieve this, the inventors first examined the labelling functionality and specificity of the HaloTag on dCas9Halo. A fluorescent HaloTag ligand, tetramethylrhodamine (TAMRA), was used to test its binding to HaloTag. The structure of TAMRA is shown in Figure 11A. Haloalkane dehalogenases is able to remove the chlorin from alkane chain by a nucleophilic displacement mechanism. The binding between HaloTag and TAMRA is highly specific, irreversible, and stable, allowing for SDS-PAGE analysis under denaturing conditions. Three batches (I, II and III) of dCas9Halo were incubated with TAMRA and analysed by in-gel fluorescence (Figure 11B). The SDS-PAGE gel was further stained by Coomassie to assess the present proteins. For batch I, II and III, the in-gel fluorescence revealed a significant band at the size of dCas9Halo, suggesting TAMRA was successfully incorporated onto the dCas9Halo protein specifically. The presence of minor bands could indicate either impurities from protein purification or degradation products labelled by TAMRA non-specifically. The shade of bands could be caused by the large amount of protein loaded on the gel, because the resolution improved when only 1 μM of protein was present (Batch II). Overall, for all batches, the dCas9-HALO band substantially reduced once pre-incubated with the HALO-reactive G4-ligands generated in WP1, demonstrating successful incorporation of the ligands on the dCas9-protein via HALO conjugation (Figure 11). Example 3 – Cellular targeting of an individual G4 (MYC) with the dCas9-G4-ligand conjugates Once the tools were generated and validated in vitro, the inventors sought to optimise conditions for ideal G4-targeting using stable cell lines expressing both the dCas9- HALO construct and the sgRNA to target the G4 of interest. sgRNA design As a proof of concept for the general strategy, the inventors focused sgRNA targeting on the promoter region of the PVT-1 gene, which is known to have several G4- structures and to be very sensitive to exposure to G4-ligands. By targeting this promoter at the different G4s present, the inventors aimed to demonstrate the validity of the invention by demonstrating that only a limited number of G4-structures present in the PVT-1 promoter were responsible for gene expression changes observed. To achieve this, the inventors generated a small library of seven sgRNAs, targeting either different G4s within the PVT-1 promoter or targeting the same structure but either within the sense or antisense strand or at the 5’ or 3’ end. sgRNA rational design is schematically depicted in Figure 12. The case of study illustrated here is focused on targeting the PVT1 oncogene due to its extensive characterisation and ability to affect gene-expression upon G4-stabilisation. This strategy may be extended to a genome-wide scale by using commercially available sgRNA libraries that will allow individual (or multiple) target of specific genomic G4s, providing the unique potential to unravel the biological relevance of individual G4 structures, which is otherwise impossible with the current available tools, and to build the fundamental biology knowledge required to exploit G4s as novel drug targets in the longer term. To investigate this and prove the validity of the invention, the inventors checked gene expression changes of PVT-1 (targeted gene) but also ELL and LRP1 genes, which crucially contains G4s but are not targeted by the dCas9-platform. Importantly, these genes are affected (in terms of gene-expression) once targeted with standard G4- ligands. Hence, the inventors’ expectation was to observe selective perturbation of PVT-1 expression (with certain sgRNA but not others) and no difference in the non- targeted genes LRP1 and ELL. Furthermore, a non-targeting sgRNA control was also added to rule out any unspecific effect that can be generated by the expression of the dCas9 when bound to a sgRNA. The results of this screening are discussed below, demonstrating selective engagement of the PVT-1 G4 with associated enhancement of gene expression. MCF7 targeting and differential gene expression changes observed only with specific sgRNAs MCF7 cell lines stably expressing sgRNA 1, sgRNA 2, sgRNA 3, sgRNA 4, sgRNA 5, sgRNA 6, sgRNA 7, and sgRNA NT, were analysed. Cells were incubated for 2 hours with 5 µM 0-PyPDS-Halo (PEG0), or 5 µM 2-PyPDS- Halo (PEG2), or 5 µM 4-PyPDS-Halo (PEG4). Cells were then washed two times with fresh media (incubated for 10 minutes after every washing), and further incubated for 6 or 18 hours prior to pellet collection and RNA extraction. As illustrated in Figures 13 to 17, gene expression changes were only observed with certain sgRNAs for the PVT-1 gene, and not with the non-targeted genes LRP1 and ELL, or the non-targeting sgRNA control. Thus, this demonstrates that the CRISPR construct according to the invention is able to selectively target specific G4s (i.e. G4s in the promoter region of PVT-1 in this example), and as a result, modify gene expression. Example 4 – Chemical labelling of CRISPR-Cas9 proteins with ligands for individual G4-targeting Catalytically inactive CRISPR-Cas9 (dCas9) fused to functional proteins have been widely used in biology to attain site-selective perturbation of gene expression. This strategy takes advantage of the selectivity provided by dCas9 bound to a short- guiding RNA (sgRNA) in recognizing a specific genomic site by base-pairing, which can be used to recruit an effector protein (i.e., a transcription factor or an epigenetic enzyme) at the targeted site. The inventors reasoned that a similar strategy could be devised to attain single G4-targeting by decorating chemically dCas9 with G4-ligands. Chemical functionalization of dCas9 proteins has been previously achieved using commercially available chloroalkane-modified fluorophores to label a dCas9-Halo fusion protein in living cells. Therefore, the inventors hypothesized that generating modified G4-ligands functionalized with chloroalkane moieties can be exploited to attain site-selective G4-targeting under physiological conditions. To achieve this, they designed analogs of the widely established G4-ligands PDS based on the previously described PyPDS scaffold. Unlike PDS, PyPDS presents a single primary amine within its structure, which can be selectively functionalized with chloroalkane sidechains by peptide coupling (Figure 18c). After successful synthesis of PyPDS following previously established methods, the inventors functionalized the primary amine of the molecule with chloroalkane side chains of different lengths, enabling systematic investigation of the ideal distance between the G4-binding scaffold PyPDS and the HaloTag protein to achieve optimal G4-engagement. Specifically, the inventors have used linkers containing different PEG repeats (n = 0, 2, 4) to vary the distance between PyPDS and the chloroalkane (Cl-PDSn, Figure 18c). To avoid limiting the use of (Approach Exact Nucleic Acid alternative structures (ATENA) to a single G4-ligand, the inventors decided to functionalize with chloroalkane moieties another widely characterized G4-ligand called PhenDC3 (Cl- PhenDC3n). Unlike PDS, PhenDC3 has a cationic side chain and is structurally bulkier, displaying a phenanthroline core wider than the pyridine one present in the PDS scaffold (Figure 18c, and d). While PDS and PhenDC3 have been extensively validated as selective G4-ligands, the structural differences between these two ligands can lead to differential biological responses when applied in cells. Therefore, the inventors decided to compare these two ligands with ATENA when recruited to a single-G4 site. To achieve this, they have synthesized a previously reported PhenDC3 analog displaying an exocyclic primary amine, which can be functionalized by peptide coupling using the same synthetic strategy described for PyPDS to afford chloroalkane-modified PhenDC3 analogs that are compatible with HaloTag conjugation (Figure 18d). With both PyPDS and PhenDC3 analogs in hand, the inventors first assessed if functionalization with the chloroalkane side chains could prevent the G4-binding properties of these molecules. To test this, they have subjected all the ligands to a standard FRET-melting assay to evaluate their ability to stabilize G4-structures. All analogs displayed good G4-stabilization, providing an increase in melting temperature (∆Tm >10oC at 4 mM) for three distinct G4 structures tested (hTelo, BCL-2 (Figure 18e) and c-KIT-2. All the chloroalkane functionalized PhenDC3 analogs showed ∆Tm values comparable to those observed with the PhenDC3, suggesting that the addition of the chloroalkane side chains had negligible effects on the G4-stabilization properties of the PhenDC3 scaffold. On the other hand, when functionalizing PyPDS with chloroalkane side chains, lower ∆Tm values than PyPDS are generally observed (Figure 18e. This agrees with previous studies demonstrating that the functionalization of the primary amine of the PyPDS scaffolds leads to a reduction of the global charge of the molecule under physiological conditions, which is associated with slightly weaker stabilization of G4-structures compared to the parent compound. Despite the reduced G4 stabilization, PyPDS analogs with a functionalized primary amine, such as SiR- PyPDS, display sufficient binding affinity and selectivity for reliable G4 detection in living cells. Having confirmed that both PyPDS and PhenDC3 analogs functionalized with chloroalkane sidechains retained good G4-binding recognition properties, the inventors next investigated if these molecules could be used to label the dCas9-Halo in vitro. To this end, the inventors expressed and purified the dCas9-Halo protein (see methods) and incubated it for 45 minutes with increasing concentrations of the G4-ligands. This was followed by incubation with an exce mM) of the commercially available HaloTag®TMR ligand to label dCas9-Halo with the TMR fluorophore. Since labeling of HaloTag is a covalent irreversible process, the inventors reasoned that if the chloroalkane functionalized G4-ligands efficiently labeled the dCas9-Halo, they would prevent the subsequent incorporation of the fluorescent HaloTag®TMR, leading to a dose-dependent reduction of TMR incorporation, as schematically depicted in Figure 18f. All the analogs tested induced a robust dose-dependent reduction of the dCas9- Halo TMR signal, demonstrating high efficiency in labeling dCas9-Halo irrespectively of the nature of G4-ligand (i.e., PyPDS or PhenDC3) or the PEG-linker used to connect the chloroalkane to the G4-binding scaffold (Fig.18g). The inventors next asked if dCas9-Halo functionalized with G4-ligands could be used to target individual G4s. To this end, they designed a dually fluorescent-labeled DNA template containing the c-KIT2 G4 forming sequence at its 3' end, which could be used to monitor G4-stabilization as a function of both the PEG-linkers and short guiding RNAs (sgRNAs) used (Figure 18h). To investigate this, they generated two sgRNAs to orient the dCas9-Halo complex towards the G4-structure sitting at either 18 (sgRNAFRET-18) or 42 (sgRNAFRET-42) base pairs from the targeted structure and whose Protospacer Adjacent Motifs (PAM) are located on the same strand of the G4 forming sequence. This is based on previous structural studies indicating that the C-terminal domain of the Cas9 protein, where the Halo protein is situated, will point towards the 3' end of the PAM sequence localized on the same strand of the targeting DNA sequence. To control for any change in G4-stability induced by forming the dCas9-Halo complex on its own, the inventors have also designed sgRNAFRET-41 that also sits at 41 base pairs from the G4 but whose PAM is located on the reverse strand, orienting the dCas9-Halo toward the 5' end of the sequence and preventing any ligand-mediated G4-engagement (Figure 18h). Moreover, the inventors have used a standard non- targeting RNA control (NTC) to rule out any potential unspecific binding that is not strictly mediated by sgRNA-driven proximity. As depicted in Figure 18i, the inventors failed to detect any significant changes in the FRET signal for any of the sgRNAs tested when the inventors decorated dCas9-Halo with Cl-PDS0. Considering that Cl-PDS0 can efficiently bind to dCas9-Halo (Figure 18g), the lack of G4-stabilization displayed by this molecule indicates that the linker connecting the PDS-scaffold to the dCas9-Halo is too short for engaging with the G4- structure. Conversely, when labeling dCas9-Halo with Cl-PDS2an increase in FRET- signal could be measured when using both sgRNAFRET-18 and sgRNAFRET-42 (Figure 18i). Interestingly, the extent of FRET changes appeared to be stronger with sgRNAFRET-18 compared to sgRNAFRET-42, suggesting th acing the dCas9-PyPDS complex closer to the G4 facilitates ligand-structure engagement (Figure 18i). Additionally, when using the control sgR, NAs (sgRNAFRET-41) no changes in the ∆FRET efficiency were detected (Fig.1i), strongly supporting that dCas9-PyPDS can induce G4-stabilization in a sgRNA dependent fashion. Similarly, when decorating dCas9-Halo with Cl-PDS4, the inventors observed an increase in the ∆FRET efficiency with sgRNAFRET-18 and sgRNAFRET-42, but to a lower extent than what was measured with Cl-PDS2, which indicated that extensively long PEG-linkers can be detrimental to G4-recognition. This demonstrated that dCas9- driven G4-ligand engagement is both molecule and sgRNA-dependent, with optimal G4-stabilization obtained when a PEG2 linker separated the PyPDS scaffold from the chloroalkane moiety and when the dCas9-Halo was placed ~18 base pairs from the targeted G4. dCas9-Halo_FRET assay Before the experiment, Cy5-3' end labeled and Cy3-5' end labeled oligos (Table S1) were annealed in 10 mM Tris HCl pH 7.5, 100 mM KCl at a final concentration of 1 μM (95 °C for 10 minutes and left for overnight cooling down at room temperature). dCas9-Halo purified protein at a final concentration of 4 μM was incubated with Cl- PDSn probes in a ratio of 1:2.5 for 45 minutes in binding buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 1 mM MgCl2,1 mM TCEP, 10% glycerol). The pretreated dCas9-Halo was then incubated with 120 pmol of purified sgRNA for 20 minutes at room temperature, followed by incubation with 200 nM of pre-annealed oligos at 37°C for 1h. The complexes were then loaded into an 8% polyacrylamide native gel and ran for 120 minutes at 4°C in 1x TBE supplemented with 2 mM MgCl2. Gels were then imaged using Typhoon FLA 9500 (GE) using the Cy3 and Cy5 filter set. Example 5 – Use of optimized ATENA for c-MYC targeting in cells Having established optimal conditions to attain dCas9-mediated G4-targeting in vitro, the inventors next sought to uncover the potential of ATENA to promote single-G4 stabilization in living cells. The use of cell lines that constitutively express dCas9-Halo is essential to ensure that the cellular levels of this protein are consistent across different experiments, avoiding bias introduced by the significantly variable levels of protein expression typically associated with transient transfection. To this end, the inventors decided to integrate dCas9-Halo into the genome of a breast cancer cell line (MCF7) using standard lentiviral (see methods), given the highly diverse response in gene-expression changes previously reported upon treatment of these cells with PDS34that could be further investigated with ATENA. Successful integration of dCas9-Halo was confirmed by PCR-based genotyping and Western Blot. Next, the inventors evaluated the ability of the chloroalkane functionalized ligands to label dCas9-Halo in cells. To achieve this, they have leveraged an established chloroalkane penetration assay (CAPA) to evaluate the relative potency of the different ligands to label dCas9-Halo in living cells (Peraro, L. et al. Cell Penetration Profiling Using the Chloroalkane Penetration Assay. J. Am. Chem. Soc.140, 11360-11369 (2018)). In short, during CAPA, cells are initially exposed to different concentrations of the chloroalkane-modified G4-ligands, which are then incubated with a Halo reactive fluorophore (Cl-Oregon Green). The efficiency of ligand incorporation can be measured as an inverse function of the Oregon Green emission, as labeling with the G4-ligands should prevent the incorporation of the fluorophore (Figure 19a). As displayed in Figure 19a, treatment of MCF7 cells stably expressing dCas9-Halo with both Cl-PDS0 and Cl-PDS4 revealed a modest reduction of Oregon Green emission, with 60% labeling obtained only at high concentrations of the ligands (10 mM). Conversely, Cl- PDS2 could label ~90% dCas9-Halo at a concentration as low as 0.25 mM, reaching saturation at 2.5 mM (Figure 19a). This suggested that the cellular permeability and bioavailability of Cl-PDS2 were particularly suitable for its application in ATENA. Encouraged by these results, the inventors initially focused on using Cl-PDS2 to achieve single-G4 targeting in cells with ATENA. The inventors started their investigation by considering the G4 present in the promoter of the c-MYC proto- oncogene, as this is one of the most studied G4 described in the literature. Many studies have linked the targeting of MYC-G4 by ligands to the downregulation of c-MYC expression. To address to what extent c-MYC expression is perturbed by ligands exclusively by stabilizing the G4 in its promoter, the inventors have designed a series of sgRNAs to guide PDS selectively at the MYC-G4 using ATENA. To ensure the correct orientation of the Halo protein decorated with Cl-PDS2 toward the MYC-G4, they designed sgRNAs targeting either the non-template strand (NT) bearing the G4- structure at its 3' end or the opposite strand at its 5' end (T) (Figure 19b). Based on their biophysical investigation, the inventors reasoned that placing sgRNAs within a widow of ~50 base pairs would have ensured G4-stabilization by Cl-PDS2tethered to dCas9 (Figure 18i). Considering PAM sequences available for dCas9-Halo binding at either G4 ends, the inventors designed sgRNAMYC-19 and sgRNAMYC-67 that would place the protein complex at respectively 19 base pairs away from the MYC-G4 at its 3' end on the T strand. Similarly, they generated sgRNAMYC+22 and sgRNAMYC+58, targeting the MYC-G4 from its 5' end and placing dCas9-Halo / sgRNA complex on the opposite strand at 22 and 58 base pairs, respectively. To further investigate the optimal distance to achieve G4 stabilization in cells with ATENA, the inventors have also designed sgRNAMYC-89 and sgRNAMYC-93, along with sgRNAMYC+75, sgRNAMYC+77, and sgRNAMYC+119, also targeting the MYC-G4 at its 3' and 5' end, respectively (Figure 19b). After cloning sequences encoding the various sgRNAs into a vector for mammal expression (see methods), the inventors have transfected MCF7 cells stably expressing dCas9-Halo with individual sgRNAs, followed by treatment with Cl-PDS2 for 48 hours. The inventors then measured changes in c-MYC expression using RT-qPCR, normalizing the expression level against individual samples transfected with the respective sgRNAs but not exposed to Cl-PDS2. This enabled them to consider any change in gene expression potentially triggered by the assembly of the dCas9-Halo complex not labeled with PDS on the promoter for each sgRNA used. To their surprise, the inventors observed that sgRNAs placing the dCas9-PyPDS complex within a ~50 bp window of the MYC-G4 on the T strand (sgRNAMYC-19, sgRNAMYC-67) led to negligible effects on c-MYC global expression (Figure 19c), which contrasts their biophysical predictions (Figure 18i). Conversely, when placing the complex on the NT strand with sgRNAMYC+22 and sgRNAMYC+58, the inventors observed reduction of c-MYC expression (~20%), which could be indicative G4-engagement mediated by ATENA (Figure 19c). To investigate this further, they placed the dCas9- PyPDS complex further away from the MYC-G4 using sgRNAMYC+75, sgRNAMYC+77, and sgRNAMYC+119, which should lead to abrogation of G4-engagement in a distance- dependent fashion. However, the inventors observed an opposite trend by which c- MYC downregulation was increased the further away the complex was from the G4 (Figure 19c), which cannot be consistent with a ligand-meditated effect. By closer inspection of the promoter annotation, the inventors noticed that sgRNAsMYC +58 to +119 overlapped with the P1 promoter and TATA-box sequences. In particular, sgRNAMYC+75 and sgRNAMYC+77 place the dCas9-PyPDS complex directly on the TATA-box sequence, which can justify the observed c-MYC suppression. This effect is exacerbated when sgRNAMYC+119, which targets the region next to the TSS of the P1 promoter, is used. Therefore, the inventors reasoned that c-MYC suppression likely reflected perturbation of the transcriptional homeostasis regulated by the TATA-box domain caused by the dCas9-PyPDS complex rather than G4-stabilization. To test this hypothesis, the inventors decorated Cl-Oregon Green or Cl-PDS2 and monitored c-MYC expression changes when using sgRNAMYC+58. Cl-Oregon Green is a fluorophore and is unable to stabilize G4s. Therefore, potential changes in c-MYC expression observed with dCas9-OregonGreen will be indicative of transcriptional dysregulation that is G4-independent. Crucially, the inventors observed a suppression of c-MYC when using dCas9-Oregon-Green that was comparable to dCas9-PyPDS (Figure 19d). This strongly suggested that targeting the MYC-G4 at its 5' end using dCas9-conjugates is unable to provide reliable observations on G4-mediated transcriptional regulation due to the interference with key regulatory sites, such as the P1 promoter and TATA-boxes. Considering that multiple promoters regulate c-MYC expression, the inventors investigated changes in transcriptional levels elicited by dCas9-PyPDS by focusing on the transcript generated by the G4-dependent P1 promoter. Indeed, it has been recently shown that deletion of the MYC-G4 from its promoter only affects the expression of c-MYC controlled by the P1-promoter, while modest perturbation of global c-MYC transcription is observed. To test this, the inventors quantified c-MYC expression changes caused by dCas9-PyPDS using primers that exclusively amplify transcripts generated by the P1 promoter. Gratifyingly, the inventors observed a distance-dependent suppression of P1-mediated c-MYC expression when targeting MYC-G4 with sgRNAMYC-19 and sgRNAMYC-67 (~6 and 4-fold, respectively, Figure 19e). Importantly, no significant changes in expression were measured when placing the dCas9-PyPDS complex further away from the G4 with sgRNAMYC-89 and sgRNAMYC-93, which is in agreement with their biophysical observations and consistent with G4- engagement (Figure 19e). Similarly, when targeting the G4 from its 5' end by placing the dCas9-PyPDS complex on the complementary strand (NT) with sgRNAMYC+22, the inventors also detected a reduction of P1-mediated c-MYC expression of 4-fold (Figure 19e). The inventors also detected some transcriptional repression when targeting G4 with sgRNAMYC+58 and sgRNAMYC+75, although this might be G4-independent effects as discussed above (Figure 19e). However, P1-mediated c-MYC expression was entirely abrogated when the dCas9-PyPDS complex was placed at sites overlapping with the GC-box and close to the P1-TSS with sgRNAMYC+119 (Figure 19e). These observations further confirmed that placing the dCas9 complex on the 5' end of the MYC-G4 cannot be used to reliably detect changes in gene expression that are strictly mediated by G4-targeting. The inventors next set out to validate if the changes in P1-dependent c-MYC expression observed upon treatment with sgRNAMYC-19 and sgRNAMYC-67 could indeed be ascribed to dCas9-PyPDS binding to the -G4. Firstly, the inventors assessed if selective targeting of the MYC-G4 with ATENA was associated with changes in the expression of another gene bearing a stable G4 in its promoter, such as KRAS. No detectable change in KRAS expression could be detected when directing ATENA to MYC-G4, which supports selective MYC-G4 targeting. Conversely, when using free PyPDS, KRAS expression is significantly reduced, further supporting how ATENA can provide intra-G4 selectivity to ligands like PyPDS. The inventors further validated ATENA-mediated G4 stabilization by looking at protein accessibility at the G4 site upon targeting. It has been proposed that ligands bound to G4s can displace key transcription factors and regulatory proteins, leading to transcription suppression. Therefore, the inventors reasoned that if dCas9-PyPDS complex was correctly positioned to enable ligand-G4 interaction, they would have observed reduced protein accessibility to the G4. To test this hypothesis, the inventors have used the G4-selective antibody BG4 and performed CUT&Tag in the presence of dCas9-PyPDS in conjunction with either sgRNAMYC-19 or a non-targeting control (NTC). They then used qPCR to measure the enrichment of the MYC-G4 signal against 3 independent validated G4-sites in MCF7 cells (MAZ, RPA3, RBBP4) to assess relative protein accessibility at these G4s under different conditions. As displayed in Figure 19f, when treating cells with dCas9-PyPDS using sgRNAMYC-19, the inventors observed a consistent reduction of BG4 accessibility that was not detected for the NTC, irrespective of the reference G4 used. This strongly indicates that PDS can bind the MYC-G4 guided by the dCas9, preventing the BG4 antibody from binding to the same target. These observations supported a model by which dCas9-PyPDS guided by accurately selected sgRNAs can lead to ligand-mediated G4-stabilization and P1- specific c-MYC downregulation. To further demonstrate that c-MYC expression changes observed with ATENA could be confidently ascribed to G4-stabilization, the inventors employed PhenDC3, as a validated G4-ligand that is structurally very different from PDS. To this end, they used the PEG-2 analog Cl-PhenDC32, which also showed good labeling of dCas9-Halo in cells by CAPA. Similarly to what was observed with PyPDS, also dCas9-PhenDC3 caused a significant reduction of P1-mediated c-MYC expression when used in conjunction with sgRNAMYC-19 (Figure 19g). Interestingly, this combination led to a higher suppression of transcription (~14-fold, Figure 19g), possibly reflecting the stronger G4-stabilization abilities of Cl-PhenDC32compared to Cl-PDS2measured by FRET. This suggested that ATENA might also be used to assess the relative intensity of phenotypes that different G4-ligands elicit on the same target. Altogether, their data indicate that ATENA can successfully target MYC-G4 in a ligand-i endent fashion, leading to detectable G4 engagement and corresponding P1-specific c-MYC suppression. Cloning of lentiviral dCas9-Halo construct The lentiviral dCas9-Halo plasmid was cloned using Gibson assembly. A lentiviral backbone containing already dCas9 (Addgene #61425) was digested using BamHI and BsrGI (New England Biolabs, R3575S, R3136S) and then assembled with the HaloTag sequence amplified (Table.S3) from pET302-6His-dCas9-Halo (Addgene #72269) in a 1:3 molar ratio of backbone: insert using HiFi DNA Assembly Mix (E2621S, New England Bioscience) following manufacturing protocols. Post incubation, assembled products were diluted with water, and 5 μL of the product was transformed by heat shock into 10-beta competent cells (New England Biolabs, C3019I). Cells were then plated on agarose plates (supplemented with ampicillin 100 μg / mL) for overnight outgrowth at 37 ℃. Single clones were picked and grown in 5 mL of Amp LB media overnight at 37 °C while shaking. Plasmid DNA was purified from cells using the Promega PureYield plasmid miniprep system (Promega, A1223) and sequenced using the Geneweiz service. The positive plasmid was further modified by substituting blasticidine resistance with mCherry coding sequence using Gibson assembly after digestion with BsrGI and EcoRI enzymes. Positive clones were then sequenced using the Geneweiz service. Cloning of sgRNA in pLG1 plasmid pLG1 backbone (Addgene #109003) was digested with BstXI and BlpI (FastDigest, ThermoFisher) for 1h at 37°C. Oligos containing the sgRNA sequence (Table S4) were ordered from IDT and annealed in water at 10 μM in a thermocycler (37°C, 30 minutes- 95°C, 5 minutes and ramp down 5 degrees / minute to 25°C). The annealed oligos were diluted (1:50) and then assembled with the digested plasmid in a 1:2 molar ratio of backbone: insert using HiFi DNA Assembly Mix (E2621S, New England Bioscience) following manufacturing protocols. Post incubation, assembled products were diluted ½ in water, and 5 μL of the product was transformed by heat shock into 10-beta competent cells (New England Biolabs, C3019I). Cells were then plated on agarose plates (supplemented with ampicillin 100 μg / mL) for overnight outgrowth at 37 ℃. Single clones were picked and grown into 5 mL of Amp LB media overnight in a 37 °C rotating shaker. Plasmid DNA was purified from cells using the Promega PureYield plasmid miniprep system (Promega, A1223) and sequenced using the Geneweiz service. Lentivirus production and transduction For lentiviral production, HEK293T cells seeded at 3.8x106in a 10 cm tissue culture plate the day before transfection.8.4 ug of the envelope plasmids pCMV-VSV- G (Addgene #8454) and 6.4 ug of packaging plasmid R8.74 (Addgene #22036) were co-transfected along with 2.1 ug of the target plasmid (dCas9-Halo-T2A-mCherry) using polyethyleneimine (PEI-Linear, MW 25000) in a ug DNA: ug PEI ratio of 1:3. Viral supernatant was harvested after 48h and 72h, and before usage, it was filtered using a 0.45 μm filter unit. MCF7 cells were plated on 6-well plates the day before infection. They were infected with lentiviruses in DMEM 10% FBS, β-Estradiol, in the presence of polybrene with a final concentration of 10 μg / mL. Half of the media was changed the day after, and cells were grown for one week and examined by flow cytometry (Attune NxT, ThermoFisher) to confirm successful transduction (YL2- Channel). After genotyping, cells were single-cell sorted using FACS BD FACS Diva 9.0.1. CAPA assay MCF7 cells stably expressing dCas9-Halo were plated the day before at 30x10^3 cells / well in 96-well plates pre-coated with Poly-D-Lysine (Thermo Fisher Scientific, A3890401). Cells were incubated for 2h in the presence of serial dilutions of Cl-PDSn or Cl-PhenDC3n probes ranging from 0.2μM to 10 μM (5% CO2, 37 °C). Cells were then washed two times with media, and every washing step included 10 minutes of incubation (5% CO2, 37 °C). Cells were then incubated with HaloTag® Oregon Green® Ligand (Promega, G2801) for 45 minutes, followed by two washing steps (10 minutes,5% CO2, 37 °C). Cells were washed with DPBS, treated with 0.25% trypsin, and resuspended in FACS buffer (5%BSA in DPBS) before flow cytometry analysis (Attune Nxt). Example 6 – Biological responses observed at specific G-quadruplexes are ligand- dependent After validating ATENA’s power in underpinning biological responses elicited by individual G4s, the inventors extended its application beyond c-MYC. Previous reports indicated that PDS treatment of MCF7 led to significant upregulation of the long non- coding RNA PVT1, which they also confirmed for PyPDS by RT-qPCR and RNA-seq. Since G4-stabilization by ligands is typically linked with transcriptional suppression, the inventors decided to deploy ATENA and investigate if PVT1 over-expression was an indirect consequence of global G4-stabilization induced by PyPDS or if it was a response reflecting the individual stabilization of the G4 present in the PVT1 promoter. Previous CUT&Tag experiments performed on the MCF7 cell line identified a G4-peak close to the PVT1 promoter, which the inventors decided to target by designing sgRNAs to position dCas9 in proximity t PVT1-G4 at either 20 base pairs from its 5' end (sgRNAPVT1-20) or 33 base pairs from its 3' end (sgRNAPVT1+33), ensuring no overlap with any regulatory region of the promoter (Figure 20a). They then transfected MCF7 expressing dCas9-Halo with sgRNAPVT1-20 and sgRNAPVT1+33 before treating with Cl-PDS2, as described earlier. In agreement with previous reports, the inventors observed a ~4-fold increase of PVT1 expression when targeting the G4 in its promoter with dCas9-PyPDS (Figure 20b). Given that gene-suppression associated with G4-ligands treatment is elicited by the displacement of key regulatory proteins from the G4, the inventors questioned whether PyPDS could instead increase protein binding to the PVT1-G4, thus leading to the observed transcriptional increase. To test this hypothesis, they have performed CUT&Tag and measured enrichment of the PVT1-G4 when targeted with dCas9-PyPDS over previously characterized G4s in MCF7 cells (MAZ, RPA3, RBBP4). Strikingly, the inventors observed that PyPDS binding to the PVT1-G4 and sgRNAPVT1-20 causes an increase of BG4-mediated enrichment of ~ 2- fold (Figure 20c), contrasting the effect that the same ligand elicits when targeting the MYC-G4 (Figure 19f). This observation suggests that the previously described increase in PVT1 expression elicited by PyPDS treatment in MCF7 cells reflects the genuine response mediated by the PVT1-G4, highlighting how varied the response of G4s can be when exposed to the same ligand. They next asked if the increase in PVT1 expression observed with PyPDS was a ligand- dependent effect or if it was indicative of a more general biological response to treatment with any G4-ligand. Interestingly, when exposing MCF7 to PhenDC3 as a free ligand not tethered to dCas9, the inventors observed PVT1 suppression rather than transcriptional enhancement. They, therefore, questioned if the response to G4- binding in the PVT1 promoter was genuinely ligand-dependent by guiding PhenDC3 selectively at this promoter using ATENA. To investigate this, the inventors have used Cl-PhenDC32 in conjunction with sgRNAPVT1-20 and sgRNAPVT1+33 to assess PhenDC3- mediated PVT1 expression changes upon selective PVT1-G4 targeting. Under these conditions, the inventors observed a suppression of PVT1 expression (Figure 20d), which is opposite to what was measured with PyPDS and in line with what was observed using PhenDC3 on its own. This suggested that G4-ligands might elicit a different response when bound to the identical G4, possibly reflecting binding modalities that can either increase or prevent protein accessibility. To further investigate ligand-mediated responses to individual G4-targeting, the inventors have synthesized a HaloTag-compatible analog of a third G4-ligand: Pyrido Dicarboxamide (PDC, Figure 20e). More specifically, they have functionalized the PCD scaffold with a PEG-2 chloroalkane side (Cl-PDC2) to mimic the PyPDS and PhenDC3 analogs used in ATENA (Figure 20e). The inventors then used Cl-PDC2 in conjunction with sgRNA-20 to target the PVT1 promoter. Similarly to what was observed with PhenDC3, PDC also caused suppression of PVT1 with a fold change of ~ 4-fold (Figure 20e), suggesting that the way PDC interacts with the PVT1-G4 is reminiscent of PhenDC3. Indeed, the PCD scaffold is similar to PhenDC3 by displaying methylated nitrogens on the quinolines that are oriented oppositely compared to PDS (Figure 20e). Moreover, both PhenDC3 and PDC lack the amino-side chains present in PyPDS, further highlighting the structural similarity between these two scaffolds. Our result indicated that the biological response elicited by ligands at individual G4s reflects the interactions between the ligand and the structure and, therefore, cannot be used as a reliable proxy to infer the endogenous function of a targeted G4 but rather should be used to infer ligand-specific responses at targeted sites. Transfection and incubation with G4 ligands MCF7 cells stably expressing dCas9-Halo were plated the day before at 25x10^3 cells / well in 96-well plates pre-coated with Poly-D-Lysine (Thermo Fisher Scientific, A3890401). According to manufacturer protocol, 50 ng of guide expressing plasmid was transfected using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001). The day after, cells were incubated with either DMSO (0.5%) or Cl-PDSn or Cl-PhenDC3n probes and incubated for 48h. RNA isolation RNA was harvested 72 hours post-transfection. Cells were washed with 100 μL of 1X DPBS (Gibco, 14190144) and incubated with RLT buffer from RNeasy mini kits (Qiagen, 74104) according to the manufacturer's instructions. Following the manufacturer's instructions, the eluted RNA was reverse transcribed to cDNA using the RevertAid cDNA prep kit (K1621, Thermo FisherScientific). Example 7 – Targeting cell-specific G4s with ATENA reveals transcriptional-dependent response to ligands CUT&Tag and other chromatin-compatible G4-mapping methods, like BG4-ChIP and Chem-Map, have revealed that G4 distribution in the genome is cell-dependent and typically prevalent at promoters of highly transcribed genes. Therefore, the inventors decided to investigate biological responses attained when directing a ligand to previously unexplored G4-structures specifically detected in MCF7 cells. In particular, the inventors wanted to investigate if the biological relevance of individual G4s and their response to ligand binding was linked to the transcriptional levels of the associated genes. To this end, the inve set out to apply ATENA to study de novo cell-specific promoter G4s, encouraged by the demonstrated ability of our method to target individual G4s within the genome of living cells. The inventors leveraged the existing dataset on G4-distribution in MCF7 cells previously obtained with CUT&Tag. This study identified a G4-peak in the promoter of the highly expressed HMGN1 gene as an exclusive marker of MCF7 cells compared to other cell lines. HMGN1 encodes for a non-histone chromosomal protein able to interact with nucleosomes and regulate chromatin structure. The inventors, therefore, questioned if they could leverage ATENA to target and stabilize the G4 in the HMGN1 promoter selectively to modulate its transcriptional level in a G4-dependent fashion. To test this, the inventors have designed sgRNAHMGN1-22 and sgRNA HMGN1+34 to target the HMGN1-G4 at 22 and 34 base pairs, respectively, at its 3' and 5' ends, ensuring no overlap with functional regions (Figure 21a). After incorporation of sgRNA HMGN1-22 and sgRNA HMGN1+34 into an expressing vector, they have independently transfected MCF7 cells with both sgRNAs, before adding Cl-PDS2 and incubating for further 48 hours, as per the optimized ATENA protocol. Strikingly, the inventors have observed an extremely strong reduction of HMGN1 expression of 76-fold when targeting its G4 at the closest distance of 22 base pairs with sgRNAHMGN-22 (Figure 21b). HMGN1 downregulation was partially attenuated when placing dCas9-PyPDS further away from the G4 with sgRNAHMGN+34, consistent with the distance-dependent ligand engagement observed for other G4s (Figure 21b). This data indicated that a G4 that is detected specifically in MCF7 cells (HMGN1) could be targeted individually with ATENA to suppress its transcriptional levels up to the higher extent observed by G4-targeting in the context of this study. This indicates that maintaining G4-homeostasis at the promoter of highly transcribed genes is key to sustaining elevated expression levels characteristic of specific cell lines, making these G4s particularly sensitive to G4 ligands. Altogether, the observations suggest that the varied transcriptional response observed upon G4- ligands treatment might reflect the relative relevance of different G4s in maintaining transcriptional homeostasis of the specific cell line studied. To corroborate this hypothesis, the inventors used ATENA to target a G4 present in the promoter region of a gene expressed at very low levels in MCF7 cells. To this end, the inventors have noticed that CUT&Tag G4-mapping performed in MCF7 cells revealed a distinct G4-peak in the promoter region of IL17RA, a gene that is only marginally expressed in this cell line. IL17RA encodes for the interleukin 17A (IL17RA), a proinflammatory cytokine secreted by activated T-lymphocytes and, therefore, not essential for breast cancer cell homeostasis. The inventors generated sgRNAIL17RA-20 to target the IL17RA-G4 at 20 base pairs from its 5' end and within a region that does not overlap with other atory elements of this promoter. After transfection with sgRNAIL17RA-20 and sgRNANTC followed by incubation with Cl-PDS2, the inventors failed to detect any measurable changes in IL17RA expression levels (Figure 21c). This indicates that targeting a G4 in a promoter of a gene that is not transcriptionally active is not associated with gene-expression perturbation, linking tightly the functional relevance of G4s to the transcriptional levels of the genes associated. Considering that both G4s in the HMNG1 and IL17RA promoters are equally detected in MCF7 by CUT&Tag and targeted with similar sgRNA designs (within the limits imposed by the PAM availability), the inventors reasoned that the transcriptional levels linked to the targeted G4 can be used to anticipate the extent of transcriptional perturbation associated with ligand treatment. This is also illustrated by the relatively modest transcriptional changes observed by the selective targeting of the c-MYC and PVT1 G4s, which are only moderately expressed in MCF7 cells. Increasing evidence in the literature suggests a model by which G4s act as epigenetic factors to mark highly transcribed genes. The data support this model by showcasing how the extent of gene suppression / activation elicited by G4 ligands is linked to basal transcriptional levels, highlighting the relevance of maintaining G4 homeostasis to sustain elevated transcription characteristics of specific cell lines. CUT&Tag-qPCR CUT&Tag libraries were diluted 1:10, and 2 µL of the diluted CUT&Tag library was mixed with 1 μM of primer mix (Table S6) and 5 μL of SYBR Green PCR Master Mix (Applied Biosystems: 4385612) according to the following protocol 20 seconds at 95 °C, 39 cycles of 10 seconds at 95 °C, 30 seconds at 60 °C, and 10 seconds at 72 °C, and finally heating to 90 °C. The Ct values obtained were used to calculate the relative fold-change in gene expression using the ΔΔCt method and assess the relative fold change at G4 target sites when compared against G4-positive regions (MAZ, RBBP4, RPA3). Summary The development of selective ligands to target DNA G-quadruplexes (G4s) has been pivotal in revealing their role in transcriptional regulation. However, most of the ligands described to date lack intra-G4 selectivity, severely limiting their potential for uncovering the biological function of individual G4s across the genome. To overcome these limitations, the inventors developed ATENA (Approach to Target Exact Nucleic Acid alternative structures). ATENA relies on the chemical modification of established G4-ligands to enable their conjugation onto a catalytically inactive Cas9 protein (dCas9) using HaloTag, allowing for the targeting of individual G4s in living cells. The inventors have systematically screened of the PEG-linkers connecting the G4-ligands to the HaloTag and sgRNA sequences to attain optimal G4 engagement both in vitro and in cells. Using optimized conditions, they leveraged ATENA to demonstrate how the selective targeting of the well-studied G4 in the promoter of the oncogene c-MYC suppresses its transcription exclusively from the P1 promoter. They also show that positioning ligands in the proximity of regulatory elements suppresses c-MYC transcription in a G4-independent manner, highlighting the importance of appropriate design to measure genuine G4-mediated transcriptional changes. They also demonstrate that selective targeting of a G4 in the PVT1 promoter can either stimulate or repress its transcription depending on the type of G4-ligand used, indicating that functional responses associated with G4-stabilization can highly depend on the type of ligand used. The inventors further harnessed ATENA to study transcriptional perturbation associated with cell-specific G4s, revealing that the functional responses associated with these structures are tightly linked with the expression levels of the targeted gene. Their study provides critical insights into G4- based therapeutic design, offering an innovative platform to investigate G4 biology with high precision. Conclusions The inventors have demonstrated that by conjugating a G4 binding ligand to a dCas9 protein, the G4 binding ligand can be positioned within the proximity of a particular G4 of interest using a dedicated short guiding RNA. As such, the inventors have developed a chemical-biology platform that can be used to target individual G4s within the human genome. The significance of this invention is two-fold. Firstly, this platform will provide immediate impact by offering a novel chemical tool with unprecedented control and resolution that can be utilised to investigate G4- biology, enabling perturbation of a single G4 at a time. More specifically, this invention will allow the investigation of the role and potential of specific G4-structures as targets for therapeutic intervention, including a variety of important human diseases, such as cancer and rare genetic diseases (e.g., Cockayne Syndrome). Notably, this cannot be achieved with current tools available. Secondly, this invention allows the generation, for the first time, of the tools to achieve chemical functionalisation of an established biological tool for gene-editing (CRISPR-Cas9), which offers limitless long-term applications stretching far beyond the study of G4-biology. Indeed, the dCas9-HALO platform described in this invention can be applied to investigate any desired DNA-interacting molecule, providing unprecedented sequence-specific recog . For example, the approach can be extended to the study of sequence-specific responses elicited by chemotherapy drugs (e.g., cisplatin or topoisomerase inhibitors) or to guide epigenetic drugs (e.g., HDAC inhibitors) onto specific genomic locations, for the very first time. This highlights the importance of this strategy and its scientific value for the study of any type of DNA- molecule interaction, offering unprecedented opportunities to investigate biological responses elicited by such interactions and to study the mechanism of action of chemotherapy drugs from a new perspective.

Claims

ms 1. A CRISPR construct comprising a G-quadruplex (G4) binding ligand.

2. The CRISPR construct according to claim 1, wherein the CRISPR construct comprises an gRNA (guide RNA) or an sgRNA and / or an effector protein.

3. The CRISPR construct according to claim 2, wherein the effector protein comprises a CRISPR-associated endonuclease protein, optionally wherein the CRISPR- associated endonuclease protein comprises a Cas protein.

4. The CRISPR construct according to claim 3, wherein the Cas protein is selected from a group consisting of: Cas1, Cas2, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas13, Csm, Cmr, and RNase III, optionally wherein the Cas protein is Cas9.

5. The CRISPR construct according to claim 3, wherein the CRISPR-associated endonuclease protein comprises a Cpf1 protein.

6. The CRISPR construct according to claim 2, wherein the effector protein comprises an endonuclease-deficient protein, optionally wherein the endonuclease- deficient protein comprises a dCas9 protein.

7. The CRISPR construct according to any one of claims 2 to 6, wherein the gRNA comprises trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA), or a single guide RNA (sgRNA), optionally wherein the gRNA comprises a sgRNA.

8. The CRISPR construct according to any one of claims 2 to 7, wherein the gRNA recognises a target DNA region in close proximity to a G-quadruplex, optionally wherein the gRNA recognises a target DNA region within at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, or at least 400 nucleotides of a G- quadruplex, or wherein the gRNA recognises a target DNA region within at least 500 nucleotides, at least 1000 nucleotides, at least 1500 nucleotides, or at least 2000 nucleotides of a G-quadruplex.

9. The CRISPR construct according to any one of claims 2 to 8, wherein the gRNA recognises a target DNA region in close proximity to a G-quadruplex, optionally wherein the gRNA recognises a target DNA region within at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, or at least 40 nucleotides of a G-quadruplex (G4), or wherein the gRNA a target DNA region within at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, or at least 90 nucleotides of a G-quadruplex (G4), or wherein the gRNA recognises a target DNA region within at least 100 nucleotides of a G-quadruplex (G4).

10. The CRISPR construct according to any one of claims 2 to 9, wherein the gRNA is not configured to recognise or target a DNA region that is coding, optionally wherein the gRNA is not configured to recognise or target a DNA region that is an intron or exon in an ORF; or wherein the gRNA is configured to recognise or target a DNA region that is non-coding.

11. The CRISPR construct according to any one of claims 2 to 9, wherein the gRNA is configured to recognise or target a DNA region within telomeric DNA, a gene expression regulation region, a promoter region, ribosomal DNA, an enhancer, or a pseudogene.

12. The CRISPR construct according to any one of the preceding claims, wherein the G-quadruplex (G4) to which the G4 binding ligand can bind is located in a gene or an oncogene, optionally wherein the G4 is located in a promoter of the gene or oncogene.

13. The CRISPR construct according to claim 12, wherein the gene is IL17RA, or the oncogene is the myc, N-MYC, PVT1, KRAS or c-KIT gene.

14. The CRISPR construct according to any one of claims 1 to 13, wherein the CRISPR construct is configured to target one or more G4s, optionally wherein the CRISPR construct is configured to target one G4.

15. The CRISPR construct according to any one of claims 1 to 14, wherein the G4 binding ligand is non-specific.

16. The CRISPR construct according to any one of the preceding claims, wherein the G4 binding ligand comprises pyridostatin (PDS), PhenDC3, CX5461, BRACO19, Pyrido Dicarboxamide (PDC), or Cl-PDC2, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, optionally wherein the G4 binding ligand comprises pyridostatin (PDS), or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof.

17. The CRISPR construct according to claim 16, wherein: (i) pyridostatin comprises Formula (I): 5 ; :: ;(V):(v) Pyrido Dicarboxamide (PDC) comprises Formula VIII:(VIII); and / or (vi) Cl-PDC2 comprises Formula (IX):(IX).

18. The CRISPR construct according to any one of the preceding claims, wherein the G4 binding ligand comprises a pyridostatin-analogue comprising pyrrolidine sidechains (PyPDS), or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof.

19. The CRISPR construct according to claim 18, wherein PyPDS comprises Formula (II):).

20. The CRISPR construct according to any one of the preceding claims, wherein the CRISPR construct further comprises a modified haloalkane dehydrogenase moiety.

21. The CRISPR construct according to claim 20, wherein the modified haloalkane dehydrogenase moiety conjugates the G4 binding ligand to the CRISPR construct, optionally the effector protein thereof, and optionally the dCas9 protein.

22. The CRISPR construct according to claim 20 or claim 21, wherein the modified haloalkane dehydrogenase moiety comprises at least one amino acid modification relative to a corresponding wildtype haloalkane dehydrogenase, wherein the modification of the modified haloalkane dehydrogenase allows a chloroalkane linker substrate to be irreversibly bound to its active site.

23. The CRISPR construct according to any one of claims 20 to 22, wherein the modified haloalkane dehydrogenase moiety comprises an amino acid substitution at a position corresponding to amino acid residue 272 of Rhodococcus rhodochrous dehalogenase, optionally wherein the substituted amino acid at the position corresponding to amino acid residue 272 is phenylalanine, glycine or alanine.

24. The CRISPR construct according to any one of the preceding claims, wherein the G4 binding ligand comprises or is functionalised with a chloroalkane moiety, optionally a polyethylene glycol (PEG)-chloroalkane moiety.

25. The CRISPR construct according to claim 24, wherein the polyethylene glycol (PEG) comprises one ethylene oxide unit, wherein the PEG comprises two or three ethylene oxide units, wherein the PEG comprises four or five ethylene oxide units, wherein the PEG comprises six or seven ethylene oxide units, or wherein the PEG comprises eight ethylene oxide units.

26. The CRISPR construct according 24 or claim 25, wherein the polyethylene glycol (PEG) comprises four ethylene oxide units.

27. The CRISPR construct according to claim 24 or claim 25, wherein the polyethylene glycol (PEG) comprises no ethylene oxide units.

28. A G-quadruplex (G4) binding ligand conjugated to a haloalkane dehydrogenase moiety.

29. A compound of formula (VI): G4-L0-L1-L2-L3-L4-Cl (VI) or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein: G4 is a G-quadruplex (G4) binding ligand; L0is absent or is an optionally substituted heteroatom; L1is absent or is an optionally substituted C1-20 alkylene, an optionally substituted C2- 20 alkenylene, an optionally substituted C2-20 alkynylene or a polyethylene glycol (PEG) group; L2is absent or is an optionally substituted heteroatom; L3is absent or is an optionally substituted C1-20 alkylene, an optionally substituted C2- 20 alkenylene, an optionally substituted C2-20 alkynylene or a polyethylene glycol (PEG) group; and L4is absent or is an optionally substituted C1-20 alkylene, an optionally substituted C2- 20 alkenylene or an optionally substituted C2-20 alkynylene.

30. The compound of claim 29, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein L0is absent.

31. The compound of claim 29 or 30, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein L1is an optionally substituted C1-10 alkylene, an optionally substituted C2-10 alkenylene or an optionally substituted C2-10 alkynylene, optionally wherein L1is an optionally substituted C3-7 alkylene, an optionally substituted C3-6 alkenylene or an optionally substituted C3-7alkynylene.

32. The compound of any one of 31, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein L1is , wherein m is an integer between 1 and 5, optionally wherein m is 3.

33. The compound of any one of claims 29-32, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein L2is absent or is O, S or NH, optionally wherein L2is NH.

34. The compound of any one claims 29-33, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein L3is , wherein n is an integer between 1 and 15, and the asterisk representsbonding to L4where it is present, or to Cl where L4is absent.

35. The compound of any one of claims 29-34, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein L4is an optionally substituted C1-10 alkylene, an optionally substituted C2-10 alkenylene or an optionally substituted C2-10 alkynylene, optionally wherein L4is an optionally substituted C3-8 alkylene; and optionally wherein L4is –(CH2)6-.

36. The compound of any one of claims 29-35, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein the alkylene, alkenylene or alkynylene is independently substituted with one or two oxo groups.

37. The compound of any one of claims 29-36, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein the G4 binding ligand is non-specific.

38. The compound of any one of claims 29-37, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein the G4 binding ligand comprises pyridostatin (PDS), PhenDC3, CX5461, BRACO19, Pyrido Dicarboxamide (PDC), or Cl-PDC2, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, optionally wherein the G4 binding ligand comprises pyridostatin (PDS), or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof.

39. The compound of claim 38, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein: (i) pyridostatin comprises Formula (I): ;;(V):(v) Pyrido Dicarboxamide (PDC) comprises Formula VIII:; and / or (vi) Cl-PDC2 comprises Formula (IX):(IX).

40. The compound of any one of claims 29-37, or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein the G4 binding ligand comprises a pyridostatin-analogue comprising pyrrolidine sidechains (PyPDS), or a pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof.

41. The compound of claim 40, or a acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof, wherein PyPDS comprises Formula (II): (II).

42. A method of targeting a G-quadruplex (G4) in a DNA sequence, the method comprising contacting a DNA sequence comprising a target G4, with the CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to claim any one of claims 29-41.

43. The method according to claim 42, wherein the target G4 is located in telomeric DNA, a gene expression regulation region, a promoter region, ribosomal DNA, an enhancer, or a pseudogene.

44. The method according to claim 42 or claim 43, wherein the target G4 is located in a gene or an oncogene, optionally wherein the target G4 is located in a promoter of the gene or oncogene.

45. The method according to claim 44, wherein the gene is IL17RA, or the oncogene is the myc, N-MYC, PVT1, KRAS or c-KIT gene.

46. A method of modifying gene expression, the method comprising contacting a gene comprising a target G-quadruplex (G4), with the CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41.

47. The method according to claim 46, wherein the target G4 is located in a promoter of the gene.

48. The CRISPR construct according one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41, for use in therapy.

49. The CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41, for use in treating or ameliorating a disease in a subject in need thereof.

50. The CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41, for use in preventing a disease in a subject in need thereof.

51. The CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41, for use in diagnosing or prognosing a disease in a subject.

52. A method of treating or ameliorating a disease in a subject in need thereof, the method comprising administering, or having administered, to the subject in need thereof a therapeutically effective amount of the CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41.

53. A method of preventing a disease in a subject in need thereof, the method comprising administering, or having administered, to the subject in need thereof a prophylactically effective amount of the CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41.

54. A method of diagnosing or a disease in a subject, the method comprising detecting a G quadruplex (G4) in a biological sample obtained from the subject with the CRISPR construct according to any one of claims 1 to 27, the G- quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41.

55. The method according to any one of claims 42-47 and 54, wherein the method is performed in vivo.

56. The method according to any one of claims 42-47 and 54, wherein the method is performed in vitro or ex vivo, optionally wherein the method is performed in vitro.

57. The CRISPR construct for use according to any one of claims 49-51, or the method according to any one of claims 52-56, wherein the disease is mediated by a G- quadruplex (G4).

58. The CRISPR construct for use according to any one of claims 49-51 and 57, or the method according to any one of claims 52-56, wherein the subject is a human.

59. A pharmaceutical composition comprising the CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41, and optionally a pharmaceutically acceptable vehicle.

60. A process for making the pharmaceutical composition according to claim 59, the process comprising combining a therapeutically effective amount of the CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof according to any one of claims 29-41, with a pharmaceutically acceptable vehicle.

61. A method of identifying and / or validating a G-quadruplex (G4) as a therapeutic target based on the expression levels of a gene of interest, the method comprising contacting a gene of interest comprising a target G4, with the CRISPR construct according to any one of claims 1 to 27, the G-quadruplex (G4) binding ligand according to claim 28, or the compound, or pharmaceutically acceptable salt, solvate,tautomeric form, stereoisomer or form thereof according to any one of claims 29-41.

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