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Us 2019 / 0264232 A1 US 20190264232A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2019 /0264232 A1 HOU et al. ( 43 ) Pub . Date : Aug . 29 , 2019 ( 54 ) NOVEL CASO ORTHOLOGS Publication Classification (51 ) Int. CI. (71 ) Applicant: PIONEER HI- BRED C12N 15 / 90 (2006 . 01 ) INTERNATIONAL , INC . , C12N 15 / 85 ( 2006 .01 ) JOHNSTON , IA (US ) C12N 15 /52 ( 2006 .01 ) C12N 15 / 113 ( 2006 .01 ) (72 ) Inventors : ZHENGLIN HOU , ANKENY, IA C12N 15 / 10 (2006 .01 ) (US ) ; JOSHUA K . YOUNG , C12N 9 / 22 ( 2006 .01 ) JOHNSTON , IA (US ) ; GIEDRIUS ( 52 ) U . S . CI. GASIUNAS , VILNIUS (LT ) ; CPC . .. .. .. C12N 15 / 907 ( 2013 . 01 ) ; C12N 15 /85 VIRGINIJUS SIKSNYS , VILNIUS ( 2013 . 01 ) ; C12N 9 / 22 (2013 . 01 ) ; C12N 15 / 113 (LT ) ( 2013 . 01 ) ; C12N 15/ 102 (2013 . 01 ) ; C12N 15 /52 (2013 .01 ) (73 ) Assignee : PIONEER HI- BRED (57 ) ABSTRACT INTERNATIONAL , INC . , Compositions and methods are provided for novel Cas9 JOHNSTON , IA (US ) orthologs, including, but not limiting to , novel guide poly nucleotide / Cas9 endonucleases complexes , single or dual guide RNAs, guide RNA elements , and Cas9 endonucleases . (21 ) Appl. No. : 16 /282 ,498 The present disclosure also describes methods for creating a double strand break in a target polynucleotide , methods for genome modification of a target sequence under various in (22 ) Filed : Feb . 22 , 2019 vivo and in vitro conditions , in the genome of a cell , for gene editing, and for inserting a polynucleotide of interest into the Related U . S . Application Data genome of a cell . Also provided are nucleic acid constructs (60 ) Provisional application No. 62/ 651, 991, filed on Apr. and cells having a modified target site or altered polynucle 3 , 2018 , provisional application No . 62 /634 , 257, filed otide of interest produced by the methods described herein . on Feb . 23 , 2018 . Specification includes a Sequence Listing . 2 . **** * ** * * *** * ** * * rew 1 CELLEN ** ht. * ** * * ** . .. V VERA aut SNAKBAR AVAT . XVVW 4:17 * * SA We Patent Application Publication Aug. 29, 2019 Sheet 1 of 23 US 2019 / 0264232 A1 FIG . 1 . .. \ XII. a * M M 9 . 2 ** * * XI. LE II. NEN * ASH **** **** * * * * * * * * * t*** * * ** ** *** a YA XXXXX HARDT AA WAN W : ** * 2 LESSIT : -* IV 9. : 4 . Decor WA 2 . : Mein 30 Patent Application Publication Aug. 29, 2019 Sheet 2 of 23 US 2019 / 0264232 A1 . 3 : 22 Patent Application Publication Aug. 29 , 2019 Sheet 3 of 23 US 2019 / 0264232 A1 N{C>D) {D96 10126 D{A>GT)AA 10136 NNNNATW NNNNNNN AAAAWIT>C) N{T>MG)ANOW N>G)BHN(H GNT>M) 1088 H[Y>A} 10125 ID131 NRGNNNNNNDATIT YYYYYYYYYY ID85 NA>B)N 0124 NRTHA (2G)/N{C>TAG NATARCNAG NNNN NNAAAGNNNAGAAA 0104 NAR NAANACNNT>MG)CC D122 0121 iiiiiiiiiiiiiiiiiiiii N{T>V)NNNNND(WGGNNA}NTM 084 INGCNGONNNNCAIN NANNNNCYAANNNNCTA D116 101011093101390981 MN(B>A)GGNNGGNWSNTVGGNGG|NGH}GGD NAAAAYN IMINARNIT>A5). 0119101201071 D(A>TG)N 083 1063 {A>C)YN(WG?NGNN FIG.3 ACN NNNNNNN NNGAM+{A^}BNNGAP 1087010910112110127013210138081 YNNNNN VINTAA>NNGACNANT WH D9 viviviviviv D35 D60 NNNNCVGN(CMO)NNT CCN INN(H>G)AANTV\NAAANAVAC8GDW JAN(G>AYNTNTCNNTCN/KC) i NNNCACN 1091 NMR NA>YG)A D33 ANN 1D56 - NATAANINVA>GC)R - NNAAACN 0681070 INGGWGNN (A>TS)N1AGACCN V )NAT>S 06 {D22 {D3Z {052 N(C>DH GHW}C> (Y>A)N{BAL N ANANANANANANANANANANANANANANANANANANANAN - NNNNCTAA NTGAR(G>A) 05 NNNNCCA, D105 019 1051 1067 N{H_GCAA NATTTTT vvvvv NIIN(A>GC)CC NNGAAANNNAAAAANEA>YG)N NV(G>AC)RNNABRN NAGNGCNNATCOIN NN(H>G)O A>B) |ANAK-CNAPT>Gc)NNAPT}{/1CG7C4 04 D103 ID18 1029 GGON)A>K 1050 A>TG)AYHITAAAANRTAANNH{YN(R ???????????????????????????????????? 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