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WO 2017/054721 Al 6 April 2017 (06.04.2017) 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 2017/054721 Al 6 April 2017 (06.04.2017) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 15/82 (2006.01) C12N 15/113 (2010.01) kind of national protection available): AE, AG, AL, AM, A01H 5/00 (2006.01) 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, (21) Number: International Application DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/CN2016/100533 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, 28 September 2016 (28.09.201 6) MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (25) Filing Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (26) Publication Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 2015 1063 1450.5 (84) Designated States (unless otherwise indicated, for every 29 September 2015 (29.09.2015) CN kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: INSTITUTE OF GENETICS AND DEVEL¬ TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, OPMENTAL BIOLOGY, CHINESE ACADEMY OF TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, SCIENCES [CN/CN]; No. 1 West Beichen Road, Chaoy- DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, ang District, Beijing 100101 (CN). 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, (72) Inventors: XIE, Qi; No. 1 West Beichen Road, Chaoyang GW, KM, ML, MR, NE, SN, TD, TG). District, Beijing 100101 (CN). YAN, Liuhua; No. 1 West Beichen Road, Chaoyang District, Beijing 100101 (CN). Published: WEI, Shaowei; No. 1 West Beichen Road, Chaoyang Dis — with international search report (Art. 21(3)) trict, Beijing 100101 (CN). YANG, Weicai; No. 1 West Beichen Road, Chaoyang District, Beijing 100101 (CN). — before the expiration of the time limit for amending the LI, Hongju; No. 1 West Beichen Road, Chaoyang District, claims and to be republished in the event of receipt of Beijing 100101 (CN). amendments (Rule 48.2(h)) (74) Agent: NTD PATENT AND TRADEMARK AGENCY — with sequence listing part of description (Rule 5.2(a)) LIMITED; 10th Floor, Block A, Investment Plaza, 27 Jin- rongdajie, Xicheng District, Beijing 100033 (CN). o o (54) Title: A CRISPR/Cas9 System for high efficient site-directed altering of plant genomes (57) Abstract: Cassettes comprising a YAO promoter operably linked to at least one nucleotide sequence encoding a nuclease, vec - tors comprising the same are provided. A system for altering a plant genome comprising a nucleotide sequence encoding a nuclease operably linked to a YAO promoter and a method to alter the target nucleic acid molecule by using the system are provided. Plants, progeny and seeds thereof having such altered target nucleic acid molecules are also provided. A CRISPR/Cfl«9 System for high efficient site-directed altering of plant genomes REFERENCE TO RELATED APPLICATIONS This application claims priority to previously filed and co-pending application CN1 05177038, filed September 29, 201 5, the contents of which are incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted in ASCII format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on September 26, 2016, is named P12040WO00_SL.txt and is 105,189 bytes in size. TECHNICAL FIELD The present invention relates to the field of biotechnology, particularly a CRISPR/ Cas9 system for high efficient site-directed altering of plant genomes. BACKGROUND The realization of high efficient, site-directed altering for plant genomes is of great significance to study the functions of plant genes. At present, gene modification techniques, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and CRISPR C etc., have been widely used in scientific research, wherein the CRISPR C x technique is a recently developed gene modification technique. The CRISPR/C system is an acquired immune system presently discovered which exists in most bacteria and all archaea to eliminate extraneous plastids or phages, and to leave extraneous gene fragments in autologous genomes as "memories". Different forms of deletions or insertions have been created at target fragments by editing organism genomes with a CRISPR/ Cas9 system, which has been successfully used in organisms such as Homo sapiens cell lines, Danio rerio, Rattus norvegicus, Mus musculus, Drosophila melanogaster etc. In the field of plants, this technique has also been used in plants such as Arabidopsis thaliana, Oryza saliva L., Zea maysL., Nicotiana tabacum, Lycopersicon esculentum etc., but the editing efficiency of the existing CRISPR C x system is low. At present, the promoters used for driving nucleases in these systems, such as the Cas9 gene expressionor Fokl gene expression are mostly are CMV 35S promoter and Ubiquitin promoter, but previous studies have demonstrated that, the editing efficiencies of Cas9 to plant genomes driven by the both are low. It can be seen that, for improving the editing efficiencies, it is especially important to select suitable promoters for driving the expression of Cas9 gene. SUMMARY OF THE INVENTION Increased frequency of gene altering is provided by use of a YAO promoter. When used with a gene editing system such as CRISPR C , TALEN or Zinc finger nucleases, the frequency of gene editing is increased compared to use of a promoter that is not the YAO promoter and in particular compared to using the 35S promoter. In one embodiment the YAO promoter is operably linked with a nucleic acid molecule that encodes a Cas9 or Fokl polypeptide. Gene editing frequency is increased to at least 75% or more and up to 90%, 95% or more. The frequency of gene editing of a targeted nucleic acid molecule is at least five times, 18 times or higher than when using a 35S promoter. The increased gene frequency is also provided in progeny of a plant into which a cassette is introduced comprising the YAO promoter driving a nuclease such as the Cas9 or Fokl nucleic acid molecule. Cassettes, vector, edited plants and cells are also provided. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A and IB is a diagram showing structure of the CRISPR C z binary vectors for Arabidopsis transformation. The hSpCas9 cassette is driven by the 35S (see Figure 1A) or YAO (Figure IB) promoter, while sgRNA is controlled by the AtU6-26 promoter. NLS refers to the nuclear localization sequence. Figure 2 is a gel showing RFLP detect the site-directed editing effects of 35S:Cas91 6-26- sgRNA system and pYAO:Cas9lAtU6-26-sgRNA system on endogenous gene BRI1 of Arabidopsis thaliana. Here, M is a DNA Marker; Lanes 1-23 in Figure 2A are electrophoresis results of PCR products of Tl generation Arabidopsis thaliana introduced with 35S:Cas9iAt 6- 26-sgRNA system after EcoR V enzyme cleavage, Lanes 1-21 in Figure 2B are electrophoresis results of PCR products of Tl generation Arabidopsis thaliana introduced with system after EcoR V enzyme cleavage; and Col-0 is electrophoresis result of PCR products of wild type Arabidopsis thaliana after EcoR V enzyme cleavage. Figure 3A-C are graphs showing sequencing analysis for site-directed editing effects of 35S:Cas9/AtXJ6-26-sgRNA system and ? :C /AtU6-26-sgRNA system on endogenous gene BRI1 of T l generation Arabidopsis thaliana. Here, Figure 3A is a peak profile of sequencing for PCR products of 35S:hSpCas9-BRIl-sgKNA system vs. 35S-6-TI ; Figure 3B is a peak profile of sequencing for PCR products ofpYAO:hSpCas9-BRIl -sgRNA system vs. pYAO- 16-T1 ; Figure 3C is a peak profile of sequencing for PCR products of pYAO:hSpCas9-BRIl - sgRNA system vs. ρΥΑΟ- -Ί \ Figure 4A Figure 4A shows editing forms of 35S-6-TI and ρΥΑΟ-\6- Ύ\ at target sites of BRI1 gene (SEQ ID NOS 75-77, respectively, in order of appearance); and Figure 4B shows editing forms ΐ ρΥΑΟ- -Ί \ at target sites of BRI1 gene (SEQ ID NOS 75, 78, 79, 77 and 80, respectively, in order of appearance); WT represents the nucleotide sequences of wild-type Arabidopsis thaliana at the target sites, "D" represents the sequences subjected to deletion mutations, "+" represents the sequences subjected to insertion mutations, and the numbers behind "D/+" represent the amount of deleted or inserted nucleotides. Figure 5 shows representative sequences of several mutant alleles of BRI1 identified from the p :hSpC s -BRIl -sgRNA Tl transgenic plant line 4 and line 2 1 (SEQ ID NOS 81-86, 83 and 87, respectively, in order of appearance). The wild-type sequence is shown at the top with the PAM sequence in bold. Figure 6A is a gel showing RFLP analysis of genomic DNA from the pYAO.hSpCas9 -PDS3- sgRNA Tl plants. Figure 6B shows representative sequences of several mutant alleles of PDS3 identified from a pYAO:hSpCas9 - O S -sgRNA Tl transgenic plant (SEQ ID NOS 88-96, 91, 94, 92, 97, 9 1 and 98, respectively, in order of appearance).
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