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WO 2017/223538 Al 28 December 2017 (28.12.2017) W !P O PCT

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WO 2017/223538 Al 28 December 2017 (28.12.2017) W !P O PCT (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 2017/223538 Al 28 December 2017 (28.12.2017) W !P O PCT (51) International Patent Classification: sey, Ibrahim; 1010 16th Street, Apt. 608, San Francisco, A01N 63/00 (2006.01) C12N 15/10 (2006.01) CA 94107 (US). C12N 1/21 (2006.01) C12N 15/11 (2006.01) (74) Agent: DIPETRILLO, Christen G. et al; Wilson Sonsi- CI2N 15/00 (2006.01) ni Goodrich & Rosati, 650 Page Mill Road, Palo Alto, CA (21) International Application Number: 94304-1050 (US). PCT/US20 17/039 146 (81) Designated States (unless otherwise indicated, for every (22) International Filing Date: kind of national protection available): AE, AG, AL, AM, 23 June 2017 (23.06.2017) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (25) Filing Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (26) Publication Langi English HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (30) Priority Data: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 62/354,5 16 24 June 2016 (24.06.2016) US OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 62/367,386 27 July 2016 (27.07.2016) us SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, 62/483,930 10 April 2017 (10.04.2017) us TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants: THE REGENTS OF THE UNIVERSITY (84) Designated States (unless otherwise indicated, for every OF COLORADO, A BODY CORPORATE [US/US]; kind of regional protection available): ARIPO (BW, GH, 1800 Grant Street, 8th Floor, Denver, CO 80203 (US). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, MUSE BIOTECHNOLOGY, INC. [ /US]; 5500 Central UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, Avenue, Denver, CO 80301 (US). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (72) Inventors: GILL, Ryan, T.; 2025 Ash Street, Denver, CO EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 80207 (US). GARST, Andrew; 5505 Valmont Road, #266, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Boulder, CO 80301 (US). LIPSCOMB, Tanya Elizabeth, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Warnecke; 5333 Euclid Avenue, Boulder, CO 80303 (US). KM, ML, MR, NE, SN, TD, TG). BASSALO, Marcelo, Colika; 3605 Table Mesa Drive, Apt. P-286, Boulder, CO 80303 (US). ZEITOUN, Ram¬ (54) Title: METHODS FOR GENERATING BARCODED COMBINATORIAL LIBRARIES Figure 3A (57) Abstract: Provided herein are methods and composition for trackable genetic variant libraries . Further provided herein are methods and compositions for recursive engineering. Further provided herein are methods and compositions for multiplex engineering. Further © provided herein are methods and compositions for enriching for editing and trackable engineered sequences and cells using nucleic o acid-guided nucleases. [Continued on nextpage] WO 2017/223538 Al llll II II 11III I II I II III I I II 11III II I II Published: — with international search report (Art. 21(3)) — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) METHODS FOR GENERATING BARCODED COMBINATORIAL LIBRARIES CROSS-REFERENCE [0001] The present application claims priority to U.S. Provisional Application Serial No. 62/354,516, filed June 24, 2016; U.S. Provisional Application Serial No. 62/367,386, filed July 27, 2016; and U.S. Provisional Application Serial No. 62/483,930, filed April 10, 2017, the contents of each being hereby incorporated by reference in their entirety. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH [0002] This disclosure was made with the support of the United States government under Contract number DE-SC0008812 by the Department of Energy. SEQUENCE LISTING [0003] This application contains a sequence list in Table 5. BACKGROUND OF THE DISCLOSURE [0004] Understanding the relationship between a protein's amino acid structure and its overall function continues to be of great practical, clinical, and scientific significance for biologists and engineers. Directed evolution can be a powerful engineering and discovery tool, but the random and often combinatorial nature of mutations makes their individual impacts difficult to quantify and thus challenges further engineering. More systematic analysis of contributions of individual residues or saturation mutagenesis remains labor- and time-intensive for entire proteins and simply is not possible on reasonable timescales for editing of multiple proteins in parallel, such as metabolic pathways or multi-protein complexes, using standard methods. SUMMARY OF THE DISCLOSURE [0005] Disclosed herein are compositions comprising: i) a first donor nucleic acid comprising: a) a modified first target nucleic acid sequence; b) a first protospacer adjacent motif (PAM) mutation; and c) a first guide nucleic acid sequence comprising a first spacer region complementary to a portion of the first target nucleic acid; and ii) a second donor nucleic acid comprising: a) a barcode corresponding to the modified first target nucleic acid sequence; and b) a second guide nucleic acid sequence comprising a second spacer region complementary to a portion of a second target nucleic acid. Further disclosed are compositions wherein the modified first target nucleic acid sequence comprises at least one inserted, deleted, or substituted nucleic acid compared to a corresponding un-modified first target nucleic acid. Further disclosed are compositions wherein the first guide nucleic acid and second guide nucleic acid are compatible with a nucleic acid-guided nuclease. Further disclosed are compositions wherein the nucleic acid-guided nuclease is a Type II or Type V Cas protein. Further disclosed are compositions wherein the nucleic acid-guided nuclease is a Cas9 homologue or a Cpfl homologue. Further disclosed are compositions wherein the second donor nucleic acid comprises a second PAM mutation. Further disclosed are compositions wherein the second donor nucleic acid sequence comprises a regulatory sequence or a mutation to turn a screenable or selectable marker on or off. Further disclosed are compositions wherein the second donor nucleic acid sequence targets a unique landing site. [0006] Disclosed herein are methods of genome engineering, the method comprising: a) contacting a population of cells with a polynucleotide, wherein each cell comprises a first target nucleic acid, a second target nucleic acid, and a nucleic acid-guided nuclease, wherein the polynucleotide comprises 1) an editing cassette comprising: i) a modified first target nucleic acid sequence; ii) a first protospacer adjacent motif (PAM) mutation; iii) a first guide nucleic acid sequence comprising a spacer region complementary to a portion of the first target nucleic acid and compatible with the nucleic acid-guided nuclease; and 2) a recorder cassette comprising i) a barcode corresponding to the modified first target nucleic acid sequence; and ii) a second guide nucleic acid sequence comprising a second spacer region complementary to a portion of the second target nucleic acid and compatible with the nucleic acid-guided nuclease; b) allowing the first guide nucleic acid sequence, the second guide nucleic acid sequence, and the nucleic acid- guided nuclease to create a genome edit within the first target nucleic acid and the second target nucleic acid. Further disclosed are methods further comprising c) sequencing a portion of the barcode, thereby identifying the modified first target nucleic acid that was inserted within the first target nucleic acid in step a). Further disclosed are methods wherein the nucleic acid-guided nuclease is a CRISPR nuclease. Further disclosed are methods wherein the PAM mutation is not recognized by the nucleic acid-guided nuclease. Further disclosed are methods wherein the nucleic acid-guided nuclease is a Type II or Type V Cas protein. Further disclosed are methods wherein the nucleic acid-guided nuclease is a Cas9 homologue or a Cpfl homologue. Further disclosed are methods wherein the recorder cassette further comprises a second PAM mutation that is not recognized by the nucleic acid-guided nuclease. [0007] Disclosed herein are methods of selectable recursive genetic engineering comprising a) contacting cells comprising a nucleic acid-guided nuclease with a polynucleotide comprising a recorder cassette, said recorder cassette comprising i) a nucleic acid sequence that recombines into a unique landing site incorporated during a previous round of engineering, wherein the nucleic acid sequence comprises a unique barcode; and ii) a guide RNA compatible with the nucleic acid-guided nuclease that targets the unique landing site; and b) allowing the nucleic acid-guided nuclease to edit the unique landing site, thereby incorporating the unique barcode into the unique landing site. Further disclosed are methods wherein the nucleic acid sequence further comprises a regulatory sequence that turns transcription of a screenable or selectable marker on or off. Further disclosed are methods wherein the nucleic acid sequence further comprises a PAM mutation that is not compatible with the nucleic acid-guided nuclease. Further disclosed are methods wherein the nucleic acid sequence further comprises a second unique landing site for subsequent engineering rounds. Further disclosed are methods wherein the polynucleotide further comprises an editing cassette comprising a) a modified first target nucleic acid sequence; b) a first protospacer adjacent motif (PAM) mutation; and c) a first guide nucleic acid sequence comprising a first spacer region complementary to a portion of the first target nucleic acid, wherein the unique barcode corresponds to the modified first target nucleic acid such that the modified target nucleic acid can be identified by the unique barcode.
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