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WO 2016/028843 A2 25 February 2016 (25.02.2016) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/028843 A2 25 February 2016 (25.02.2016) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/68 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (21) International Application Number: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, PCT/US20 15/045805 DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 19 August 2015 (19.08.2015) KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (25) Filing Language: English PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (26) Publication Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 62/039,341 19 August 2014 (19.08.2014) US (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant: PRESIDENT AND FELLOWS OF HAR¬ GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, VARD COLLEGE [US/US]; 17 Quincy Street, Cam TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, bridge, Massachusetts 02138 (US). TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors: CHURCH, George M.; 218 Kent Street, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, Brookline, Massachusetts 02446 (US). VIGNEAULT, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Frederic; 103 Sudbury Road, Ashland, Massachusetts GW, KM, ML, MR, NE, SN, TD, TG). 0172 1 (US). MIR, Kalim U.; 30 Elmwood Avenue, Cam bridge, Massachusetts 02138 (US). Published: (74) Agent: IWANICKI, John P.; 28 State Street, Suite 1800, — without international search report and to be republished Boston, Massachusetts 02109 (US). upon receipt of that report (Rule 48.2(g)) (54) Title: RNA-GUIDED SYSTEMS FOR PROBING AND MAPPING OF NUCLEIC ACIDS Figure 6A < i i ∞ 00 © (57) Abstract: Methods of detecting, probing, mapping and directed sequencing of target nucleic acids are provided using a guide o RNA and a Cas9 protein. Methods for detecting the binding of the guide RNA/Cas9 complex to a target nucleic acid where the guide RNA includes a 3' tail sequence that can hybridize to a probe are provided. Methods for detecting the binding of the guide RNA/Cas9 complex to a target nucleic acid where the complex is physically detected are provided. RNA-GUIDED SYSTEMS FOR PROBING AND MAPPING OF NUCLEIC ACIDS RELATED APPLICATION DATA This application claims priority to U.S. Provisional Patent Application No. 62/039,341, filed on August 19, 2014 and is hereby incorporated herein by reference in its entirety for all purposes. BACKGROUND Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs in complex with Cas proteins to direct degradation of complementary sequences present within invading foreign nucleic acid. See Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature All, 602-607 (2011); Gasiunas, G , Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America 109, E2579-2586 (2012); Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012); Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic acids research 39, 9275-9282 (2011); and Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annual review of genetics 45, 273-297 (2011). A recent in vitro reconstitution of the S. pyogenes type II CRISPR system demonstrated that crRNA ("CRISPR RNA") fused to a normally trans-encoded tracrRNA ("trans-activating CRISPR RNA") is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA. Expressing a gRNA homologous to a target site results in Cas9 recruitment and degradation of the target DNA. See H. Deveau et al, Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology 190, 1390 (Feb, 2008). Various uses of CRISPR/Cas9 systems are known. See WO2014/099744, WO2013 176772, US 8,697,359 and Sternberg et al., Nature, Vol. 507, pp. 62-67 (2014). SUMMARY Aspects of the present disclosure are directed to a method of detecting a target nucleic acid sequence including the steps of contacting the target nucleic acid sequence with a guide RNA sequence having a portion complementary to the target nucleic acid sequence and a Cas9 protein, wherein the guide RNA and the Cas9 protein co-localize to the target nucleic acid sequence to form a complex, and wherein the complex is detected thereby detecting the target nucleic acid sequence. According to one aspect, the method is performed ex vivo, i.e. in vitro, such as within a vessel or on a substrate. According to one aspect, the guide RNA and Cas9 proteins are prepared and isolated to be used as reagents in the in vitro methods of the present disclosure. Aspects of the present methods include the probing, such as analytical probing or preparative probing, detecting, labeling, mapping and sequencing of nucleic acids, such as DNA. For example, the present disclosure is directed to methods of probing DNA, such as at the single molecule level, for the purpose of identifying the presence of the DNA, probing DNA for the purpose of affinity purifying the DNA, mapping the DNA, to mark out specific regions of importance along the DNA, or to create sequencing start sites. According to methods described herein, a complex is formed including a guide RNA, a DNA binding protein, such as a Cas9 protein, and a double stranded DNA target sequence. According to certain aspects, DNA binding proteins within the scope of the present disclosure include a protein that forms a complex with the guide RNA and with the guide RNA guiding the complex to a double stranded DNA sequence wherein the complex binds to the DNA sequence. This aspect of the present disclosure may be referred to as co-localization of the RNA and DNA binding protein to or with the double stranded DNA. In this manner, a DNA binding protein-guide RNA complex may be used to form a detectable complex at a specific target DNA sequence, thereby detecting the presence of the target DNA sequence. According to certain aspects, the complex may be detected due to the presence of a detectable label. According to certain aspects, the complex may be directly labeled or indirectly labeled. According to certain aspects, the detectable label may be present on the guide RNA, the Cas9 protein or the complex. According to certain aspects, a colocalization factor for the guide RNA may not be a DNA-binding protein. A reagent may be used to colocalize with the guide RNA at the target nucleic acid sequence. According to certain aspects, the guide RNA need not require the presence of a DNA binding protein to be useful in certain aspects of the present disclosure. The DNA binding protein may be absent. For example, a guide RNA may itself bind to the target nucleic acid sequence and the guide RNA may have a label or other functional moiety attached thereto so as to localize the label or other functional moiety at or near the target nucleic acid sequence. According to certain aspects, the complex may be detected by detecting the structure of the complex without having a detectable label. The physical structure of the complex is probed as opposed to visualizing a fluorescent or other visually or spectroscopically detectable moiety. According to certain aspects, the complex may be detected by detecting the physic-chemical property of the complex, such as electrostatic charge, without having a detectable label. Such methods include detecting the complex using nanopore detection methods, electron microscopy, optical microscopy, scanning probe microscopy, atomic force microscopy, cantilever detection methods, quartz crystal detection methods, field effect transistor detection methods, all of which are known to those of skill in the art. One of skill will readily envision other methods which are capable of detecting the structure of the complex based on the present disclosure. According to certain aspects, the term "guide RNA" in the context of a CRISPR Cas9 system is known to those of skill in the art and includes a portion, such as a 20 nucleotide portion, that is complementary to a target nucleic acid. Methods of designing guide RNA are well known to those of skill in the art. Methods described herein include contacting the target nucleic acid sequence with a plurality of guide RNA sequences, each having a portion complementary to the target nucleic acid sequence. Methods described herein include contacting a plurality of target nucleic acid sequences with a plurality of corresponding guide RNA sequences, each having a portion complementary to a corresponding target nucleic acid sequence. According to certain aspects, guide RNA according to the present disclosure includes a portion complementary to a target nucleic acid and a 3'-tail portion or sequence which is or may be complementary to or otherwise binds to a probe sequence or detectable label.
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