AMENDMENTS TO THE CLAIM! Application Serial No. 13/842,859 Attorney Docket No. 0084443-000001 Page 2 This listing of claims will replace all prior versions and listings of claims in the application. LISTING OF CLAIMS: 1.164. Canceled. 165. (New) A method of cleaving a nucleic acid comprising contacting a target DNA molecule having a target sequence with an engineered and/or non-naturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated (Cas) (CRISPR-Cas) system comprising a) a Cas9 protein; and b) a single molecule DNA-targeling RNA comprising |) a targeter-RINA that hybridizes with the target sequence, and ji) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides, wherein the single molecule ONA-targeting RINA forms a complex with the Cas protein, whereby the single molecule DNA-targeting FINA targets the target sequence, and the Cas8 protein cleaves the target DNA molecule.Application Serial No. 13/842,859 Attorney Docket No. 0084443-000001 Page 3 166. (New) An engineered and/or non-naturally-ocourring Type Il Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated (Cas) (CRISPR-Cas) system comprising a Cas9 protein or a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, and a single molecule DNA-targeting RNA or a nucleic acid comprising a nucleotide sequence encoding the single molecule DNA-targeting RNA; wherein the single molecule DNA-targeting RNA comprises i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides, and wherein the single molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein, whereby hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas@ protein to the target DNA molecule, 167. (New) The system of Claim 166, wherein the nucleic acid comprising a nucleotide sequence encoding the Cas9 protein and/or the nucleic acid comprising a nucleotide sequence encoding the single molecule DNA-targeting RNA are one or more vectors.Application Serial No. 19/842,859 Attorney Docket No. 0084443-000001 Page 4 168. (New) A method of cleaving or editing a target DNA molecule or modulating transcription of at least one gene encoded thereon, the method comprising contacting a target DNA molecule having a target sequence with an engineered and/or non-naturally-occurting Type Il Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated (Cas) (CRISPR-Cas) system comprising: a) a single molecule DNA-targeting RNA comprising i) targeter-RNA that hybridizes with the target sequence, and ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the targeter-RNA and the activator-RNA are covalently linked to one another with intervening nucleotides; and b) a Cas9 protein, wherein the single molecule DNA-targeting RNA forms a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule, whereby said target DNA molecule is cleaved or edited or transcription of at least one gene encoded by the target DNA molecule is modulated 169. (New) The method of Claim 168, wherein the single molecule ONA-targeting RNA is transcribed from a vector comprising a nucleotide sequence encoding the single molecule DNA-targeting RNA. 170. (New) The method of Claim 168, wherein the Cas9 protein is transcribed and translated from a vector comprising a nucleotide sequence encoding the Cas9 protein. 171. (New) The method of Claim 169, wherein the nucleotide sequence is operably linked to a control element operable in a desired celll type. 172, (New) The method of Claim 170, wherein the nucleotide sequence is operably linked to ‘control element operable in a desired cell type.473. 174. 178. 176. 177. 178. 179. 180. Application Serial No. 18/842,859 Attorney Docket No. 0084443-00000 Page 5 (New) The method of Claim 169, wherein the vector is selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors. (New) The method of Claim 170, wherein the vector is selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors. (New) The method of Claim 173, wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. (New) The method of Claim 174, wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. (New) The method of Claim 168, wherein the CRISPR-Cas system comprises two or more single molecule DNA-targeting RNAs. (New) The method of Claim 168, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a non-homologous end Joining (NHEu) repair mechanism, thereby editing the target DNA molecule. (New) The method of Claim 168, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the target DNA molecule, thereby editing the target DNA molecule. (New) The method of Claim 168, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).181 182. 183. 184. 185. 186. Application Serial No. 13/842,859 Attorney Docket No. 0084443-000001 Page 6 (New) The method of Claim 180, wherein the one or more PTO(s) aid in traversal of an organelle membrane. (New) The method of Claim 180, wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. (New) The method of Claim 168, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain. (New) The method of Claim 168, wherein the Cas9 protein is transcribed and translated from a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, wherein the nucleotide sequence is modified relative to a corresponding wild-type nucleotide sequence, wherein the modification replaces one or more codons in the wild- type nucleotide sequence with one or more different codons encoding the same amino acid. (New) The method of Claim 168, wherein said Cas9 protein comprises an activity portion having an amino acid sequence that is modified compared to an amiino acid sequence of a corresponding wild-type Cas9 protein and cleaves only one strand of DNA. (New) The method of Claim 185, wherein the Cas9 protein is transcribed and translated from a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, wherein the nucleotide sequence is modified relative to a corresponding wild-type nucleotide sequence, wherein the modification replaces ane or more codons in the wild- type nucleotide sequence with one or more different codons encoding the same amino acid.187. 188. 189, 190. 191. 192. 193. 194. 195. Application Serial No. 19/842,859 Attorney Docket No. 0084443-000001 Page 7 (New) The method of Claim 185, wherein the single molecule DNA-targeling RNA is transcribed from a vector comprising a nucleotide sequence encoding the single molecule DNA-targeting RNA. (New) The method of Claim 185, wherein the Cas@ protein is transcribed and translated from a vector comprising a nucleotide sequence encoding the Cas9 protein. (New) The method of Claim 187, wherein the nucleotide sequence is operably linked to a control element operable in a desired cell type. (New) The method of Claim 188, wherein the nucleotide sequence is operably linked to a control element operable in a desired cell type. (New) The method of Claim 187, wherein the vector is selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors. (New) The method of Claim 188, wherein the vector is selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors. (New) The method of Claim 191, wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. (New) The method of Claim 192, wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. (New) The method of Claim 185, wherein the CRISPA-Cas system comprises two or more single molecule DNA-targeting RNAS.196. 197. 198. 199. 200. 201. 202. Application Serial No. 19/842,859 Attorney Docket No. 0084443-000001. Page 8 (New) The method of Claim 185, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule. (New) The method of Claim 185, wherein the method comprises editing the target DNA molecule by a homology-directed repair mechanism. (New) The method of Claim 185, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).. (New) The method of Claim 198, wherein the one or more PTD(s) aid in traversal of an organelle membrane. (New) The method of Claim 198, wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 185, wherein the Cas9 protein comprises one or more. (New) The method of Cl mutations in a RuvC domain and/or a HNH domain. (New) The method of Claim 185, wherein the Cas9 protein comprises a mutation selected from D10A and H840A with reference to the numbering of Streptococcus pyogenes Cas9 protein.Application Serial No. 13/842,859 Attorney Docket No. 0084443-00000+ Page 9 203. (New) An engineered and/or non-naturally occurring Type Il Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated (Cas) (CRISPR-Cas) system comprising a) a Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said Cas9 protein; and b) a single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA; wherein the single molecule DNA-targeting RNA comprises: i) a targeter-RINA that is capable of hybridizing with a target sequence in a target DNA molecule, and ii) an activator-RNA that is capable of hybridizing with the targeter-ANA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides; and wherein the single molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule, whereby said system is capable of cleaving or editing the target DNA molecule or modulating transcription of at least one gene encoded by the target DNA molecule. 204. (New) The system of Claim 203, wherein one or both of the nucleic acids of a) and b) are one or more vectors and wherein the nucleotide sequence encoding said Caso protein and/or the nucleotide sequence encoding said single molecule DNA-targeting RNA are operably linked to a control element operable in a desired cell type. 205. (New) The system of Claim 203, comprising two or more single molecule DNA targeting RNAs, or one or more nucleic acids comprising a nucleotide sequence encoding single molecule DNA-targeting RNAs.206. 207. 208. 209. 210. att 212. 213, Application Serial No. 19/842,859 Attorney Docket No. 0084443-000001 Page 10 (New) The system of Claim 203, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s). (New) The system of Claim 206, wherein the one or more PTD(s) aid in traversal of an organelle membrane (New) The system of Claim 208, wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. (New) The system of Claim 204, wherein the one or more vectors are selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors. (New) The system of Claim 209, wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. (New) The system of Claim 203, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain (New) The system of Claim 203, wherein the nucleotide sequence encoding said Cas9 protein is modified relative to a corresponding wild-type nucleotide sequence, wherein the modification replaces one or more codons in the wild-type nucleotide sequence with one or more different codons encoding the same amino acid. (New) The system of Claim 203, wherein said Cas9 protein comprises an activity portion having an amino acid sequence that is modified compared to an amino acid sequence of a corresponding wild-type Cas9 protein and is capable of cleaving only one strand of DNA. 10214. 215. 216. 217. 218. 219. 220. 221 222. Application Serial No. 13/842,859 Attorney Docket No. 0084443-000001 Page 11 (New) The system of Claim 213, wherein the nucleotide sequence encoding said Cas9 protein is modified relative to a corresponding wild-type nucleotide sequence, wherein the modification replaces one or more codons in the wild-type nucleotide sequence with ‘one or more different codons encoding the same amino acid. (New) The system of Claim 213, comprising two or more single molecule DNA- targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs. (New) The system of Claim 213, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)). (New) The system of Claim 216, wherein the one or more PTD(s) aid in traversal of an organelle membrane. (New) The system of Claim 216, wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. (New) The system of Claim 213, wherein the Cas9 protein comprises one or more mutations in a Ruv domain and/or a HNH domain. (New) The system of Claim 213, wherein the Cas9 protein comprises a mutation selected from D10A and H840A with reference to the numbering of Streptococcus pyogenes Cas9 protein. (New) The system of Claim 220, wherein the Cas9 protein comprises the mutation D10A. (New) The system of Claim 220, wherein the Cas9 protein comprises the mutation H840A, 1223. 224, 225, ‘Application Serial No. 13/842,859 Attorney Docket No. 0084443-000001 Page 12 (New) A single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises: i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and il) an activator-RINA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex of a protein-binding segment, ‘wherein |) and i) are arranged in a 5'to 3' orientation and are covalently linked to one another with intervening nucleotides; wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas@ protein and hybriaization of the targeter-RINA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule, and ‘wherein the single molecule DNA-targeting RNA comprises one or more sequence modifications compared to a sequence of a corresponding wild type tracrRNA and/or rRNA. (New) A Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising the single molecule DNA-targeting RNA of Claim 223 and a Cas@ protein, (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 223, wherein the modification comprises an engineered secondary structure. 12226. 227. 228. 229, 230. 231. Application Serial No. 13/842,859 ‘Attorney Docket No. 0084443-000001 Page 13 (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 223, wherein the modification comprises a reduction in the length and/or degree of complementation in a region of hybridization, compared to a region of hybridization of a wild type DNA-targeting RNA, in the portion of the protein-binding segment that forms a double-stranded RNA duplex (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 223, wherein the protein-binding segment comprises an artificial loop. (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 223, wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433. (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 223, wherein the modification comprises adding, removing, or otherwise altering loops and/or hairpins in the single molecule DNA-targeting RNA. (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 223, wherein the modification comprises one or more additional segments at the 5" end of the targeter-ANA. (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 223, wherein the modification comprises one or more modified nucleotides in the nucleotide sequence. 13232. 233. 234. 235, 236. 237. Application Serial No. 19/842,859 Attorney Docket No. 0084443-000001 Page 14 (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RIA, of Claim 231, wherein the one or more modified nucleotides comprises at least one non-naturally- occurring nucleotide, nucleotide mimetic, or analog thereof. (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 232, wherein the one or more modified nucleotides are modified at the ribose, phosphate, andior base moiety. (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 232, wherein the one or more modified nucleotides are selected from the group consisting of 2-O-methyl analogs or 2tluoro analogs. (New) The single molecule DNA-targeting RNA, or the nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, of Claim 282, wherein the one or more modified nucleotides are selected from the group consisting of 2-aminopurine, §-bromo-uridine, pseudouridine, and 7-methylguanosine. (New) A method of cleaving or editing a target DNA molecule or modulating transcription of a gene encoded by the target DNA molecule, the method comprising contacting the system of Ciaim 224 with a target DNA molecule having the target sequence. (New) The method of Claim 236, wherein the target DNA molecule is edited, wherein the editing comprises modifying the target DNA molecule by inserting a sequence of a donor polynucleotide, said insertion resulting in an insertion, deletion, or substitution of one or more nucleotides. 14238. 239. 240. 241. 242. 243. 244. 245. Application Serial No. 13/842,859 Attorney Docket No. 0084443-000001 Page 15 (New) The method of Claim 168, wherein said Cas9 protein comprises an activity portion having an amino acid sequence that is modified compared to an amino acid sequence of a corresponding wild-type Cas9 protein. (New) The method of Claim 168, wherein the activator-RNA comprising the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433, (New) The method of Claim 168, wherein the Cas9 protein comprises a mutation selected from D10A and H840A with reference to the numbering of Streptococcus pyogenes Cas9 protein. (New) The method of Claim 240, wherein the Cas9 protein comprises the mutation HB40/. (New) The method of Claim 168, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule, (New) The system of Claim 203, wherein said Cas9 protein comprises an activity portion having an amino acid sequence that is modified compared to an amino acid sequence of a corresponding wild-type Cas9 protein. (New) The system of Claim 203, wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433. (New) The system of Claim 203, wherein the Cas protein comprises a mutation selected from D10A and H840A with reference to the numbering of Streptococcus pyogenes Cas9 protein. 15246. 247. Application Serial No. 13/842,859 ‘Attorney Docket No. 0084443-000001 Page 16 (Now) The system of Claim 245, wherein the Cas9 protein comprises the mutation He40A. (New) The system of Claim 203, wherein the system comprises a donor polynucleotide and the system is capable of editing the target DNA molecule by inserting a sequence of the donor polynucleotide into a cleaved strand of the target DNA molecule. 16IN THE UNITED STATES PATENT AND TRADEMARK OFFICE, In re application of: Confirmation No. 8182 THE REGENTS OF THE UNIVERSITY OF Examiner: NOT ASSIGNED CALIFORNIA et al Technology Center/Art Unit: 1636 Application No.: 13/842,859 Filed: March 15,2013 THIRD PARTY SUBMISSION UNDER 37 CFR. $ 1.290 For: METHODS AND COMPOSITIONS FOR RNA- DIRECTED TARGET DNA MODIFICATION. . Customer No.: 115985 Commissioner: The Examiner is requested to consider the following remarks regarding: WO 2013/141680 (cite no. 1) as they relate to pending claims 1, 2, 5-8, 10-19, 22- 25, 29-35, 38-45, 48-52, 56, 58-60, 64, 65, 73-77, 84, 86, 89, 102-106, 109-111, 113-121, 148, and 149 in US 13/842,859 (the '859 application); Sapranauskas er al. (cite no, 2) as they relate to claims 1, 2, 6-8, 11, 1214-17, 38, 40, 44, 45, 49-52, 56, 64, 65, 74-77, 109-118, and 121 in the '859 application ; Gameau er al. (cite no. 3) as they relate to claims 1, 2, 6, 11, 44, 45, 49- 56, 64, 65, and 74-77 in the '859 application; Notice of Allowance and Fee(s) for US 14/054,414 (cite no. 4) as they related to claims 38, 40, 41, 43, 64, 69, 70, 92-95, 98-102, 104, 122, 130, and 131 in the '859 application; Mali ¢7 al. (cite no, 5) and Cong er al. (cite no, 6) as they relate to claims 38, 40, 64, 69, 70, 92-95, 98-102, and 104 in the ‘859 application; WO 2014/065596 (cite no. 7) as they relate to claims 38, 40, 41, 43, 64, 69, 70, 92-95, 98-102, and 104 104 in the *859 application; and, WO 2014/089290 (cite no. 8), US 8,697,359 (cite no 9), and WO 2014/099750 no, 10) as they relate to claims 38, 40, 41, 43, 64, 69, 70, 92-95, 98-102, 104, 122, 130, and 13 in the '859 application. The remarks cite to the paragraph numbering of the '859 publication, US 2014- 0068797 Al (the "797 publication).Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page? WoO 2013141680 (Cite No.1 CLAIM t ‘WO 2013/141680 Claim 1 As scusedon p. 23, ns 1630, WO 2013141680 discloses NAsrrRNA compl iin cope of DNAageing RNAS seed ADNAsupeing RNA comping in claim and defined at [0136] & [0137] ofthe "97 publition. ‘The DNA rgting RNAS st fat in WO 2013/4168 ince many of he same element discussed inthe peiication ofthis pplication, (i) aft segment comprising «ule segue tt complementary oa sequence na target DNA and DNA arging RNAS having segment conplemenay oa sequence na ‘target DNA of elim | iene dslosed a. Fgue 20C and tp, 2, lines 79a pe 24, ines 1-28 of WO 2013140 Figure 20 (iscusd on, ines 14.28 discloses a crRNALacRNA copes having a paver egion complementary oa sue ina target DNA and. ines 7-9 discloses [ interne gis phage end plaid DNA wove by therapies CRISPR3 runes te presence, within the trget DNA, ofa rotospuoersouenoe complementary tothe pared rRNA? ia seond segment that inkras waste dicted mdiling polypoid DNAsagetng RNAS having a cond segment tatters wth te- diet ming polyepie of cain |isexpesydsloedat eg Figure 20 of WO) 201314680, which ses eRNA.trceRNA compled wih Oss, “Site dread motifing poppies wed inca Lief in paragraph [O18] ofthe 797 pubation inclu Ca. Cain The DNA tating RNA of cli |, wherein he fist egmen comprises 8 ruckoides tt have 1 complemen a equ in th tget DNA, The fist segment comprises ® mci tat ave 10% complement to a sagen inthe tet DNA ofl is dled ate, ci 24 of WO 2013/4168 wc ceted oe protspacer sequence ina target DNA tat is 10% complementary ta 20-mcetide spacer equnce ina GRNA. Chaim $ The DNAtapeting NA of cin |, when tested moijing olypepide compris en nina acid equnc having teas about 759% WO 2013141680 cisloses DNA-trgeting RNAs having at east about 75% anno cds guen: ident oth conesgnting potions of SEQID NO:Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page} WO 2013/141680 (Cite No. 1 CLAIM ‘WO 2013/141680. ‘amin acid sequence ety to amino acs 7-166 31-103 C9 ain acd sequence deen 1G 3 0 tothe coresponding tons in ny ofthe amino acid sequences set orth as SEQ ID NOs I-56 and BEM, 1317. SEQUDNO: [in WO201374 680 as rete tn 107 iio ‘SEQID NO: 1317 in claim §, Claim 6 ADNA polynckoide comprising a mule eee tht ene the DNA-eting RNA of in 1 The DNA plynucetide ending 2 DNA-trging RNA of claim 6s islet a eg, page, ines 2-24 and Fi. LB of WO 201314168, which isles pCa) has sequencing encoding DNAagetng INA (vacRNA ad SP) Chaim 7 recombinant expression etrcuprsingte DNA polymeleie of claim 4 The eonbinant epeson vector comping aDNA poljrckide cncing a DNA-rgting RNA of lai Tis lose seg, pines 2- Mand Fig. [of WO 201341680 which dla cheat eresetation fifrlogous iin two plasmids use farthe aversion of the Cas8- crRNA complex pCas SP isan expression vector with souenoes noting 4 DNAHtreting RNA (ra RNA ad SPI), Chaim 8 The reombinant expression etr of cm 7, ween the aloe sequence encoding the DNA tretng RNA i operably linked toa prom, The vcr in wich te aukoide eqeneeeoing te DNA geting BNA isoperty ike a promote of cin 8 isso at 3 ies 30.32 and claim 17 of WO 201/14168, wich dose ht the components ofthe Cas complete (ineuing ar RNA and erRNA) en be in plasmids containing bt promote), Claim 1) The reoombinan! expresso eir of chim 7, when th uli sequent econ th DNA-tgtng RNA of cai fuer comprises 2 ull loring si, The vectors iter conpsng mull cloning sites of claim 101 isosed a, 25. fines 17.22 of WO 201314168, wich dsr the cloning of CaSPI psi, Chim 17 Ain vito genetically modified host cel omprisng the DNA plynuclie The geticlymolied hs cel comping DNA polytuleoties noting DNA ering RNA of chi 1s ck at pI ies 3.27 of WOApplication No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Paged WO 2013/141680 (Cite No. 1 ‘WO 2013/141680. of claim 6, 141680, which discloses how the pCasi-)SPI plasmid was introduced into E.coli cells, ‘Chim 12 resin expression et comprising: WO 213761680 dso reconbiartexeson vero comprising racleotde sequences encodings DNA tein RNA anda sie dited ding polypeptide. Se, eg, .3ines 3032, wich disclose thatthe components fhe Cas compeesincting Cs), racrRNA, and rRNA) canbe iin sng pls. Sea 9.29 (claim 17, (Tena segue ending DNA ring RNA, when DNA- ‘aretng RNA comprises: 2) fit segment comprising a mucleotide sequence ‘atiscomplemenry toa sence ina tare DNA; nd) 2 second segment hat inka wha ste- dred ming polyp and ‘WO 2013141680 does uke sequences encoving DNA tating RNAs. Se dssson facia, (ianuckaide equne enoning the ste-ieted modifing olypeptibe comprising: aan RNA-binding prion hat inert with the DNA targeting NA, and (nativity potion hat existe dee enya ati, herein he site of enymati atvityis deterine bythe DNA-tgetng RNA TWO 2013418 dls ecnbinan exesion eos comprising rote sequen ening Cs in atone nuckeie sotence cnowinga DNAirgtig RNA (eg, cRNA and tracrRNA). “Sie dese ming poypepli” as wed in cli 12s defied in US 214 M6STST io incude Cad. Claim 13 A revonbinat expression vector omprnng: ‘WO 2013/141680 discloses recombinant expression vectors, See discussion for claim 12, above, (Tanai seguro endings DNA ring RNA, when heDNA- ‘ewig RNA copies 2 fis seen comping mcletie sequence ttatiscomplemeniry toa sence inate DNA; and) 2a segment hat interes waste dred ming polyp and WO 2013/41680 dco rexombinantexpeson vcs with ule sequences ening DNA geting RNAS, Se discussion for cai 2 aoe (ive ruckoteseqpne eng te se dieed modifi ope, heen sede ming polpeide compris: (2) an RNA- binding portion that interacts withthe DNA-tretng RNA; nd (ban activity gorio tt mois rnsriionwitin heft DNA, when the se of modulated transcription within the target DNA is determined by the DNA- WO 21374168 dsloses olenide sequences encoding se decd uahng polypepies that ave an RNA-binding portion at ners with DNAdaptng DNA and an att portion tat modus transcription within te age DNA, Seep, 1 ines 23, which ists Cas) mutes in which th endaeanyrbonuckase acy ised he mutans canApplication No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 PageS WO 2013/141680 (Cite No. 1 ‘WO 2013/141680. ving RNA lave ly one stand sie of bh sands) Vacant Cs st-ieted polyps eli redued endodeoyrbomukase atte infued an exangl of sided nding polpepties wih navy portion that mules arson of target DNA in paragraph [0409] of US 201410068797, Chaim 1 A variant site ieted mailing polpeplide comprising (Daa RNA inking portion tira whe DNA ageing RNA, ‘be DNA trptng RNA comprises aac sequence hat is complementary ia eqn ina target DNA and (i) anactivty potion hat exhibits roducedserected enzymatic activity, vein te si of ext activity i dein hy th DNA rting RNA. Wo 2013141680 ciscloes variant se reced mating polypeptides. See discussion for claim 13, above, Chaim 15 ‘The variant steed modifying polypeptide of cam 14, comprising an S404 matation oe. pyogenes sequence SQ NOS orth conespnting maton in any of te anno acd sequen se fthasEQID ‘NOs:1-256 and 795-1346. WO 23161680 does variant sie rected modi comping a coreg tion n SEQ ID NO Lines 23-2 (closing Cas mutants with tied HN aie temo, exemplified by NI91A and 868A). These mutations ge he same ative site (HNH) as HRAUA, ‘The unmutatd Cas) soquence in WO20L3 141680 (SEQID NO: 1) is 10% identical in quence SEQ ID NO: 31 inci 13. Claim 16 The aint se dred mong pole of cm 4 comprising DIOA mutton oft. pyogenes sec SEQ ID NO athe comesponding mutation in any ofthe amino aid sequenses set fot s SEQ ID Nos 26 and 95.1346, WO 213141680 dloes vat ste deed moilying poppies comprising acoresponing mtn in SEQ ID NO: 1317, Se, eg, page 3, Lines 23-2 (closing Cas mata with muted Ran ative site mi exemplified by D31A), Th D31A muti tages the same ate site (RuvC)asDI0A. ‘The unm Cas) soquence in WO20L3 141680 (SEQ ID NO: 1 i 10%Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Pages WO 2013/141680 (Cite No. 1 ‘WO 2013/141680. iden a sguence to SEQID NO: 1317 ncn I. Claim 17 17 The var sidered moda polypeptide of clin 4, comprising bath WO 2013141680 ciscloes variant screed moaving polypeptides. See discussion for claims 15-16, above, (De DIGR maton of eyo sequence SEQID NOR ie conespnting maton in any of te anno acd sequen se fthas EQ ID ‘NOs:1-256 and 795-1346; and WO 2013141680 dloes vat ste ted moda polypeptides faving tations coreponing to DIDA. See discon for cai 6 above (Gyan RDA mutton oe S,pyogns sequence SEQID NOS ort comespening mutation in any ofthe anno acd sequences seth as SEQID NOs 286 and 951346, WO 2013147680 discloses varia ste directed modlyng polypepies faving tations corespening to HBAIA, Se dscson fe claim 15, abe Chaim 18 ‘A cher se dieted nding polypeptide comprising (Gan RNA Finn potion tat incr wit a DNA arpeing RNA, when the DNAtargtng RNA comprises anceidesequne hati complementary ia equne ina tage! DNA; and (ian activity potion tates dietdeazymati activity, wherein these ofenzmati atts deine bythe DNA targeting RNA WO 2314168 does chinersite-deed moi ying polypeptide, See, eg, ll nes 12 (een steed Cs and Hind Cas9), See also discussion for claims | and 12, above. “Chiner polyp” df in paragraph 0115 of US 201406897 a include polypeptides made by the combination isin of to obese scare seen of anno acids. Claim 19 The chime sired ning polyp of cai 18 compesing en amino aid sequen having tes about 75% ain aid segue identity to amino wis 71660" 731-1018 ofthe Cs9 Cs anno ac sequene depicted in 1G. 3 orto the corespoting ports in ayo tai aid sequen sf 8 SEQ ID NOs 286 and 95.134, \W0 20131468 dls chines se deed nding poy avingat est 75% eqns ident o oto mor of he SEQID NOs in claim 19. See discussion for claim 5, above, ‘Chim 22 ‘The chimeric ste ined modiyng polyepide of lai 18, wherein he eosyatic activity modes the tut DNA, ‘WO 2013/141680 discloses site-directed modifying polypeptides with aya ay ht mites tet DNA, Se, 9.23, ies 31.32, and (p.24, lines 7-24 (disclosing how Cas9-crRNA complexes cleave both strandsApplication No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 PageT WO 2013/141680 (Cite No. 1 ‘WO 2013/141680 af rg DNA ate ses) ‘Claim 23 The chime sided ning polypeie of din 2, wherein te earynatic att is ruckase acy, methane ety, demety ase, activity, DNA npuiraiviy, DNA damage cy, deamination att, cistae avy, alain avy, depuration avi, oiatin ati, pyvinidn dime forming atv, integrase avy, tansposis acti evonbie avy, polymers ati, ie atviy, bela atviy, phos ati or gieospae acy. ‘WO 2013/141680 discloses chimeric site-directed modifying polypeptides with nuclease activity, See discussion for claim 22, above Chim 2 The chimeric stedinedmodiyng polypepide of lai 23, wherein he enzymatic activity is nuclease atvity. WO 2013/4160 cisloses chimeri sie-recied malying polypeptides with nuclease ati, See discussion far claim 2, ave Chaim 25 The chime seine lng polyeide of din 24, wherein te rules at inte double ste bein th target DNA. Wo 2013/4680 ciscloes chimeric sie-reced mealying polypeptides with double strand nuclease activity. See discussion for claim 22, above Claim 29 ‘An RNA polymuclide comprising 2 ruled sequence encoding the chimeric eed modiljng polypeptide of im 1, ‘WO 2013141680 discloses RNA comprising a muclvide sequence encodings cher site-directed moilfngpolypepi,Se, eg, pI nes 7-12 (disclosing step aggod Cas) and éxBistaggd Cas). RNA is necessary an ferme sp in pring he Cs) pron om he pASKIBA3 Cs) les orth pBAD Cas pls Claim 3 ‘ANA pljuckotide comprising alee eee encoding the chime site-deved mying ply of cin 1, 7 WO 20031660 dls DNA peracid ecdng chines ied lying polyps Se sus or cin 9, hove. Claim 31 A resoinant expression elor comprising te plymuletibeof im 3, ‘WO 2013/141680 discloses expression vectors comprising DNA polynucleotides encoding chimeric site-lirecied mifying polypeptides. SeeApplication No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 8 WO 2013/141680 (Cite No. 1 ‘WO 2013/141680. «iscassion fr claim 29, above, ‘Claim 32 The oben expression vert clin I, wherein he poynaties openly Hn apron ‘WO 2013)/141680 discloses expression vectors comprising DNA rely ening chine sired odin olpeptites, whith pluses nko promo See dscusin ein 29, above. Claim 4 Ania vio ently mdf ist cell comprising the plyncetie of claim 30 7 WO 20134160 dls eal mold cel ompsnga DNA oly ecg a chimeric seseted moding poppe. Se, gpl ies 3.2 (dkxng geeticaly mie Ecol el comping less exesng Cas, Claim 35 chimeric stedeed malin olpepie comprising (ax RNA ining portion fat inert with a DNA argeing RNA, wherein the DNA arptng RNA comprises amide sequence hat is complementary ta equnce ina ret DNA, end (i anactviy portion tat modulates taneiion within te txt DNA, vente seo mde tans win te txt DNA is celermied by the DNA taretng RNA. WO 23141680 dsloes cinerisie-dresad nding polypeptides. Seecisusion or clin 13 and 18 ahve Claim 38 A geaclly mdf cel omprsngeeobiant sie etd modifi polypeptide comprising an RNA hndng portion tt inert witha DNA- tei RNA; nd nai prtion tat exbissedietd engi activity, when te sie of erzymati activity determine bythe DNA- ‘eng RNA WO 2013141680 discloses genetically modified hs cel comprising DNA polynucleotide encoding a recombinant site-dievted modifying polypeptide. Se, eg. pl ines 3-27 (sling genetically moti’ Ecol els comping pls exesng Cis). ‘See also discussion for claim 12, above. Chain 39 The gncticaly moe cell of claim 38, wherein the stedieted moifving ‘Wo 2013141680 cisloses maf cells with site-directed modifyingApplication No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Paged WO 2013/141680 (Cite No. 1 CLAIM ‘WO 2013/141680. polypeptide compries en amin aid sequence ving es cout 5% amino aid sequence ety to amino ci 7-66 cr 3-108 of ti asl amin acid equnce dict n 1G 3, or the omespoding porios in any ofthe amin acid sguences st arth 5 SBQUD NOs 256 and S136, elpeties avn atest 75% amino ai sqoence ety tone ore cof the SEQ ID NOs in claim 39, See discussion for claim 5, above, Claim 40 ‘The genetically modified cel of claim 38, wherein the cel is selected from the oup consising of: an archaea cel hater cell, a eukaryote cel a clan anal el in invert cel verter cela sh celhafrgcel aid el a mammalian el piel, cow el gat cl, a sheep cel, a rodent cell, a rat cella mouse cel, a non-human primate cel, anda hun cel ell organism, a somatic cl, or cel a tem cell plat ‘WO 2013/141680 discloses E.coli cells. See discussion for claim 38, above, Claim 41 A ragenicnor-bunan organi wins geome comprises ansgene comprising a uct sequen encodings ein! ste diet odin polypeptide comping {an RNA ining portion eras wit a DNA engin RNA; and (inactivity potion tt exis sede emma activi, erin these ofenzmati atts dlerined bythe DNAcargting RNA WO 2013/4160 cisloses transgenic E ol cel, Se discussion for ims 12 and 38, above, Chim 22 The tangeni organism of clin, when the site-ieed modifying polypepide compris en amino acid seguence having st es about 3% amino aid sequence ety tain ci 7-166 cr 3-108 o th Casal ain acid equnce depicted n FG, 3, or the comping orion inany ofthat acid sequences set fora SEQID NOs 256 an B14, WO 2013141680 discloses sit rected odin polypeptides having at {east 5% identity to one or more ofthe SEQ ID NOs in claim 42, See dicusion for claim 5, above, Chaim 43 Te ranger rio of ci 1, when the anion seed on ‘WO 2013/141680 discloses transgenic E.coli, See discussion for claim 38,Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 10 WO 2013/141680 (Cite No. 1 CLAIM ‘WO 2013/141680. ihe wou omsitng of an az, haven, ekayoie sige nga, an alga plant, an anna, an invert, fa wom, rian vert afk fg bil anal, an ung, rk & fe mouse and nonhuman priate, above, Chaim A camposton opr WO 23141680 does composton comprising DNA-agting RNAS and site-dneced modifying polypeptides. Se, 2.23 ines 1622 (demorstating comple comprising Cas, RNA, and raiRNA), Se ao 28cm}, (i) a DNA targeting RNA, or DNA polymucleoide enonding the same, te DNA-treting RNA comprising (ast segment comprising aclie sequence tt compemenary ia seen in art DNA; and) 2 send semen hat ina Wise deve non pope nd ‘WO 2013/141680 discloses DNA targeting RNAs, See discussion for claim |, above, (i ese ded ying pol pede, or apolaleid enodng te sane te ede ming polypepide comprising (2) an RNA-binding eotion tht interacts with the DNA tareing RNA; an (b) an atv ption hates sere enzymatic activi, when te sof enya axis determine bye DNA tagetng RNA. \WO 20131468 dls steed mong polpepies, See ciao fr cans | nd 12, above, ‘Chim 45 The composition ofan 4, wherein te ist segment of th DNA aging NA comprises 8 mcletdes that ave atest 10s complementary oa sequence inthe tpt DNA, WO 2374168 doses composton comprising DNA-agetng RNAS and sie-dneed odin polyenes, wherein the fir set fhe DNAagtng RNA bs 100% complementary toa sequace in he gst DNA, Ser, ep 30 (lim 24) Caio $8 The composition of cin 4, wherein the ie etd nding polpepide compris an ain acid sues having! ast shot 7% nina acid sequence iden toaming ais 7166 731-11 of he Cash Cs ang ‘cid seer depicted in 1G, 3, orth oman prions nny the ain aid eguanes et fos SEQD NOs 256 and 795-1346, WO 20151468 dls steed modying pobpepieshvig at let 15% sequence ety toner o te SEQ ID NOs in chim $8 Seeisasion fr cin ato.Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Pagel WO 2013/141680 (Cite No. 1 CLAIM ‘WO 2013/141680. Claim 49 The composition of cin 44 wherein te enzymatic actly mos he tay DNA 7 WO 200314168 discloses enzymatic activity tat mies ret DNA through double strand breaks, See discussion for claim 22, above, Chaim 50) Te conposton ofan 4, wii te ent acy aks ax, mettre activity, deme aiiy, DNA reir, DNA dane ay, dant tivity dsm ctv, alkylation acy, depuration avi, oxidation activi primi dimer oning any, ilerase acy, assay, reombinse avy, polyesters ay, poly atiyor lynsey. ‘WO 2013/141680 discloses enzymatic activity that modifies target DNA. ‘rough double stand beats, Se disusion or claim 22, above. ‘Chim 31 The conpostion of cin 5, wherein te enzyatc ait is mulease ati. Wo 2013/4680 discloses enzymatic att tht moe trgt DNA. ‘through double strand breaks. See discussion for claim 22, above, Chim 52 The composition of ci I, wherein fe muck atv intodues dub strand brent get DNA, WoO 2013141680 cisloses enzymatic activity tht mois target DNA ‘through double strand breaks, See discussion for claim 22, abywe, Claim $6 The composition of im 4, wherein the DNA areng RNA fsadbl- molecule DNAtargeting RNA andthe composition comprises bh 2 tareter- RNA and ancl RNA the dpe ming sean f which re complementary and yt frm he second seen ote DNA- ‘targeting RNA, ‘WO 2013/141680 discloses double-mokecule DNA-targeting RNAS, See casio fr clam above. Chaim $8 A conposion comping {DDNA ageing NK of ci ora DNA plc encoding he same and WO 23761680 dos compostons comprising « DNA-argeing RNA ana sbiliing bull. Se p23, ines 4-28, shoving amie of Cus, crRNA, and taerRNA in but. Se as dscsion orci 44 core,Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 12 WO 2013/141680 (Cite No. 1 CLAIM ‘WO 2013/141680. (i bute for sizing mci ais, Claim 59 ‘AN composton comprising (te sited modiyng polypepide of cm 4, ra pyaucetide enon te some ad (ia herr sbiliring mackie acids andor pris WO 2013/6168 dios compostons comprising a ste diet ming rlypeptiea a sailing bull, Seep, nes 4-8, showing a mite of Ca cRNA, and taerRNA inate, Seals dsason forclaim 44, abo Chaim 6 ‘A conposon comping (DNA ring RNA ora DNA purl ending he sane, ie DNAtupeting RNA comprising: (ist semen comprising a ruckotde seqoence tit complementary i sequence ins gt DNA; nd) 2 second semen hat interac wiha sede ng pope and (i este deed lying pol pede, ora polyale enoding te sane, the sie-dreed odin plypepide comping: (2) an RNA Hndng orion ht iets wit the DNA tarting RNA nd) an vty pron ‘bat moles tars wii te ere DNA, wen ke ste of module tansspion within te tget DNA is eine byte DNA- tugeig RNA. WO 2013141680 dloss compostons comprising « DNA-areing RNA, anda tit mdihing polypeptide that modules tension within thet DNA. Se discon for cans 13 nd, hove Chim 6f Amst ose pce modisation oars DNA te metod comprising contacting the get DNA wit {DNA -argtng RNA, ora DNA pljncelbeencoding he sane, ‘hein th DNA rei RNA comp (2 fis semen comping ruck sequence tts complemen inthe tet DNA; and) ond set hikers wih ase dete modifying polypepiea (ipa sie-Tecedmoljingplpepie, ora pluton te same, when thst modi poypepiecompis (2) an RNA- WO 20131468 dloese matt see mtn of tpt DNA comprising ccoing th target DNA wih a DNAreig RNA and siete modingplypeptite Se, eg, 9.28 (ci I), See asop. 24 tins 7-14 (emonstating sie specication oa DNA doplex by nit vi assembled CasrRNA comple), Se ao discs foci 4, aboApplication No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 13 WO 2013/141680 (Cite No. 1 CLAIM ‘WO 2013/141680 Binding prio tt inact wit fe DNA-areng RNA; ed () an city portion tat exhibit diel enzymatic acti, Chaim 65 The method of claim 64, wherein the target DNA is extacbromosoma ‘WO 2013/141680 discloses extrachromosomal target DNA. See discussion for claim 64, hove, The DNA duplex iextracromnsomal Chim 73 Themed of cam 4, wri fe DNA-odiing pol pepe compris an amino ai sequence avingal st chat 73% amino ac sequence ‘identity 0 amino acids 7-166 ot 731-1003 of the Cas9\Csnl aminoacid seqence depicted in FIG, 3, ofthe eoregoning portions in any ofthe amino acid sequences set forth as SEQ ID NOs: 1-256 and 795-1346, Wo 2015141680 doses DNA sniyng polypepdes having at ast 758% sequence identity oon or nae athe SEQ ID NOsin lim 73, See RNA) and anacivatr RNA (ie, texrRNA), Sach mmpostion ae inherent preset for example, inthe colcls crying pCRISPR closed on, 927, co, st rege Claim 04 Amend of sie-specic mditcation of tet DNA, te etd comprising contacting te target DNA wit Spank eal dloes methods or sitespctic modification oa ret DNA eg. p8P pls DNA) by coneng the tre DNA witha DNA- targeting RNA anda ste-dreved modilying polypepide. See p.9280 (“We ‘show here that CRISPR3\Cas module cloned into E.coli is functionally active and provides st liners aun plasmid and pn”) (a DWAtrng RNA, ora DNA poljaucetde eooding he ane, ‘when te DNA argting RNA compris (2) first segment comprising a rule sequence tats complementary a sequence in th tg! DNA; and (ba send erent ht interacts witha ste iret modilying polypepieend Sapna dcloes DNA targeting RNAS. See discussion or chim J,ahoe, (istered mol plypepide, or polyrucide neding te same, when thst deve modifing polypeptide compris: (2) an RNA- binding portion ta inert wih he DNAating RNA; and (6) an acviy orion ht existed enya ati. Sapraaskaset al dls ste eed modihng oles. See discussion for claim 1, above, Chim 65 ‘The method of claim 64, wherein the target DNA is extrachromosomal, ‘Spraasla eal dls target DNA thas extacnensomal, For example, pSPl/pSP2 plasmid DNA an phage mia DNA (ue, 9. 9278) bsh examples of extairomnsomal DNA, Chim 14 The method of cain 64, enzymatic activity modes ae Claim 75 Sapraaskas tal dls enzymatic tite edb stand aces eetvits)ht my tuget DNA, See cso for chins 4952, abe,Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 26 ‘SAPRANAUSKAS et al, (Cite No, 2) CLAM ‘SAPRANAUSKAS etal. The metnd of clan 4 when he enzymatic acy sue acti, ety acy, dete atvity, DNA rei atity, DNA damage aviv, deamination att, dims att, alkylation ati, ¢epurinion avy, oxidation ati, pyridine dime forming atv negra atv, rerspsise avy, eombitase acviy, polymerase activity, igase atv, bela avy, photolyase ato cose actly Chaim 76 Themed of cain 75, wherein ie DNA- nding enc acy uclease activity. Claim 17 ‘The method of claim 76, wherein the nuclease activity introduces double stand breakin te tert DNA ‘Claim 109 Akitconpeing (the DNA ageing RNA of dim I, ora DNA ply ceoding he saa (Gj acaent fr econsiuon end dition Claim 110) The kit of clam 19, ier comprising reagent selected rom by oop consisting of br for nroduing int el the DNS targeting RNA, 2 sash buf, contol eget, acon exesion vec or RNA, polyno, areget or tanstng the DNA-arting RN frm DNA, and combinations thet Sapraaskas eal provides plaids encoding DNA ting RNAS angen fr dion rules fr ntdton intel, See "Paid ‘rasformatios ection pacing pp. 927677, disclosing aCe solution for inti th CRISPRS plasmid it oh Cains 111-18, 21 See discussion for claims 109-110, above,Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 27 Chim GARNEAU ea (ameau eal disses DNAareing RNA, See alo p67, co. ADNAMretng RNA comprising (Gsosing how “the CRISP Cas stem tags itherinvang DNA oc RNA” and how this occurs through CRISPR RNAs (crRNASs}). (ais seenent comprising aru equne tt i complanentry toa sequence ina target DNA; and Garrat. ses DNA get RNAs tae completly toa sequence ina target mck ai. Se 67 co. Caspr the RNAS to tage! foreign RNA by complementarity). (i). ond segment thinkers wi se etd mdling polypeptide. Garneau al sos DNA ring RNAs Tat neat wih sede odin plypeices. Se p67, | (‘Cas protis use th er RNA o tet eign RNA by complement’). “Sie decd modiyng polyp” as wd incl |is dfnd in US 201410068797 inde Cas) (se prareph 0188). Cas and Cs ae sh ames for Cash. Chaim? ‘The DNA-targeting RNA of claim 1, wherein the frst segment comprises ruck tt have complementary sequen inthe tet DNA, amen adios DNA ugeting RNA bang segment bang 10% omplemetry to pNT1 target DNA, See. 67 cl 2: Sequence anajsisof CRISPI nth elorenentoned 30 clos denied 14 fren spacers (435 al of which were enologus to pNTL sequen (able | and Fg.” Chim 6 ADNA polyno comprsinga mle een tht ene he DNA-tagting RNA of ei | Garneau ta. dsdoses DNA polauclenies ending DNA urging RNAS (eg, cRNAS. Se pg 67) Sequence of CRISPI inthe afemestioned 30 clones iden eet spaces (85-85) al of hich were bomnlogns to NT guns (Table and Fig 1)” Chim 11 Ani vi ently modified ist el comprising th DNA plynucetie of cline, Gently moi el comprising the DNA ening the cRNA were created when the pNTLvetorwaslerparced int te thermos cel Se poe 67. The intoduction ofthe pNTI vex geeialy of th bast cel Claim 4