HARNESSING HETEROLOGOUS AND ENDOGENOUS CRISPR-CAS MACHINERIES FOR EFFICIENT MARKERLESS GENOME EDITING IN CLOSTRIDIUM

摘要

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property :::` , MD HIM 011101011M 010 1111 1 0 I 0111111011010111100 111E0 ill OEN Organization International Bureau (10) International Publication Number 03 (43) International Publication Date .....•""- WO 2017/190257 Al 09 November 2017 (09.11.2017) WIP0 I PCT (51) International Patent Classification: (71) Applicant: NEEMO INC [CA/CA]; 237 Barton St. E., C12N 15/09 (2006.01) Cl 2N 15/31 (2006.01) C5-103-DBRI, Hamilton, Ontario L8L 2X2 (CA). C12N 1/21 (2006.01) C12N 9/22 (2006.01) C12N 15/00 (2006.01) C12Q 1/68 (2006.01) (72) Inventor; and C12N 15/11 (2006.01) (71) Applicant: CHUNG, Duane [CA/CA]; 237 Barton St. E., C5-103-DBRI, Hamilton, Ontario L8L 2X2 (CA). (21) International Application Number: PCT/CA2017/050805 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, 04 July 2017 (04.07.2017) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (25) Filing Language: English HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (26) Publication Language: English 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, 62/330,195 01 May 2016 (01.05.2016) US OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, 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. (54) Title: HARNESSING HETEROLOGOUS AND GENOME EDITING IN CLOSTRIDIUM FIG. : By this invention, heterologous CRISPR-Cas9 systems has revolutionized genome editing across CRISPR-Cas9 system in Clostridium Clostridium, we also explored the prospect of co-opting work was bolstered from the observation that plasmids address this barrier and establish CRISPR-Cas system. In required for site-specific nucleolytic attack. LESS C sequences 1 Type I CRISPR-Cas interference (Clostridium difficile) BpGer I Casio Cascade] , N)--PAM sequences "" waillEMEDEED — ENDOGENOUS CRISPR-CAS tracrRNA RNA pre-crRNA Case Interference for high-efficiency bacterium Clostridium all domains of life. Here we report implementation of the heterologous Type for markerless genome editing. Since 74% of species harbor CRISPR-Cas loci host-encoded CRISPR-Cas expressing heterologous cas9 result in poor transformation of we focus on characterization and exploitation of the in vivo interference Introduction of a synthetic CRISPR array and iffialEMWA MACHINERIES FOR EFFICIENT MARKER- Type II CRISPR-Cas interference (Streptococcus pyogenes) r.P::or c) , I 41 '''' ttactRNA if, RNase III '''' RNase III i ;, _PAM sequences ',swil l )11 " 11 41 laRNA / Cas3 Target recognition ,,,crRNA Cas9 1 DN =,...5 , 5 , 53uMMT i mssasrs.... DB ,.. Invader Invader C for the first time, a method systems, in the anaerobic pasteurianum proof-of-concept, silico spacer analysis and site-specific genetic engineering, utilizing either native pasteurianum, is provided. Application of CRISPR- machinery for genome editing. Motivation for Clostridium. C. pasteurianum Type assays revealed three protospacer adjacent motif (PAM) cpaAIR gene deletion template I - 2 0 (57) ...°L + or IN Cas9 kr) II el © in ON this Il --..... To IN I-B Il N C [Continued on next page] WO 2017/190257 Al MIDEDIMOMOIDEIRMEMOMOHM0111100ERVOIMIE (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, 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, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). 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)) — with information concerning request for restoration of the right of priority in respect of one or more priority claims (Rules 26bis.3 and 48.2(b)(vii)) — with sequence listing part of description (Rule 5.2(a)) yielded an editing efficiency of 100%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only of the 25% total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans. To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium. of genome editing through harnessing native CRISPR-Cas machinery in Clostridium. This is the first report