(19) *EP003011035B1* (11) EP 3 011 035 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/63 (2006.01) C12N 15/10 (2006.01) (2006.01) (2006.01) 13.05.2020 Bulletin 2020/20 C40B 40/08 C12N 9/22 (21) Application number: 14738672.6 (86) International application number: PCT/US2014/041790 (22) Date of filing: 10.06.2014 (87) International publication number: WO 2014/204723 (24.12.2014 Gazette 2014/52) (54) ASSAY FOR QUANTITATIVE EVALUATION OF TARGET SITE CLEAVAGE BY ONE OR MORE CRISPR-CAS GUIDE SEQUENCES TEST ZUR QUANTITATIVEN BEWERTUNG DER ZIELSTELLENSPALTUNG DURCH EINE ODER MEHRERE CRISPR-CAS FÜHRUNGSSEQUENZEN TEST POUR L’ÉVALUATION QUANTITATIVE DU CLIVAGE DE SITES CIBLES PAR UNE OU PLUSIEURS SÉQUENCES GUIDES DU SYSTÈME CRISPR-CAS (84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • GAJ THOMAS ET AL: "ZFN, TALEN, and GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO CRISPR/Cas-based methods for genome PL PT RO RS SE SI SK SM TR engineering", TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, (30) Priority: 17.06.2013 US 201361836123 P vol. 31, no. 7, 9 May 2013 (2013-05-09), pages 12.12.2013 US 201361915397 P 397-405, XP028571313, ISSN: 0167-7799, DOI: 10.1016/J.TIBTECH.2013.04.004 (43) Date of publication of application: • JANSSEN K P ET AL: "Mouse models of 27.04.2016 Bulletin 2016/17 K-ras-initiated carcinogenesis", BBA - REVIEWS ON CANCER, ELSEVIER SCIENCE BV, (73) Proprietors: AMSTERDAM, NL, vol. 1756, no. 2, 25 November • The Broad Institute, Inc. 2005 (2005-11-25), pages 145-154, XP027829577, Cambridge, MA 02142 (US) ISSN: 0304-419X [retrieved on 2005-11-25] • Massachusetts Institute of Technology • B. 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The government has certain rights in the invention. 15 BACKGROUND OF THE INVENTION [0003] Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by 20 allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator- like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome. 25 SUMMARY OF THE INVENTION [0004] The CRISPR-Cas system does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target. Adding 30 the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significantly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. To utilize the CRISPR-Cas system effectively for genome editing without deleterious effects, it is critical to understand aspects of engineering, optimization and tissue/organ specific delivery of these genome engineering tools, which are aspects of the claimed invention. 35 [0005] There exists a pressing need for alternative and robust systems and techniques for sequence targeting with a wide array of applications. Aspects of this invention address this need and provides related advantages. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence. 40 [0006] In one aspect, the disclosure provides methods for using one or more elements of a CRISPR system. The CRISPR complex provides an effective means for modifying a target polynucleotide. The CRISPR complex has a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucle- otide in a multiplicity of cell types in various tissues and organs. As such the CRISPR complex has a broad spectrum of applications in, e.g., gene or genome editing, gene therapy, generation of cell and animal model systems, drug 45 discovery, drug screening, disease diagnosis, and prognosis. [0007] Aspects of the disclosure relate to the CRISPR-Cas system having the utility of engineering primary cells that recapitulate the genetic complexity of human disease. This complexity arises from the interaction of multiple genetic lesions, which is particularly true in the case of proliferative disorders like cancer. [0008] The disclosure comprehends a method of inducing a proliferative condition in a an organism comprising: isolating 50 a first population of cells from the organism, transducing the first population of cells with a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the poly- nucleotide sequence comprises (a) three or more guide sequences capable of hybridizing to three or more target se- quences in genome of the organism, (b) a tracr mate sequence, and (c) a tracr sequence, and II.