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    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 Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 28 A compton comping Garneau al diss compostons comprising DNA-ugeting RNAS eg, crRNAs ands mdilyng polyenes eg, Cas pr. Se 1.67cok | sing bow “the CRISPRCa ste gt iter invading DNA or RNA" and explanng bow Cas roti ws thc RNAS i tpt feign uk acidsby conplnent). See also p, 70 (“Overall we have established that the S. thermophilus CRISPRI Caste caves bth aceriotage and planid DNA ins Ts endonuclease acy, wc sens toe ous, prosper spec an orton depend CasSisambername for Ci), Se, eg, Cite No.2: Sepranaskas eal p 26,1 (a (amen nae aad exe) (a DNAtareng RNA, ora DNA polyruclide enondig the sane, te DNAstgting RNA comprising (ais seanent comprising a racket sequence tas complementary oa sequence ina target DNA; and) sezond segment thinkers wt iirc mang poled nd areata. dsdoses DNA-agtng RNAs, See dun foci above, (i) the site-iexted modifying polypeptide, o a polynucleotide encoding the sang, the sie-dreced modi plypepide comprising (2 an RNA tndng porin tha nerats wih the DNA arting RN; and (9) an act potion ‘ht exits site diet enzymatic att, wherein sito enzymatic activity detemind byte DNA geting RNA Ganau etl dhs seid lying poppies, Sedan fori, sbve. Caio 45. The compsition of ci 4, wherein he ist egment of the DNA rgting NA comprises 8 nocletdes that ave atest 10s complementary oa sequen inthe tug! DNA, area a. discloses segmenting 10% complet tre DNA. Session for eli above Chim 49 The compton of ain 4, when te enya avy modes the ret DNA, Claim $0) Garneau ta diss wonpostion He toe nein that kave endomuclesatit that mdf taget DNA though double tand breaks, Seep 70(*Overl we have ests tht he. thermopias CCRISPRUCas jt cles oth uteri nd plaid DNA in vi Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 29 GARNEAU tal Cie. Te conpostion fain 4, wherein fe enzyme metas city, deme activi, DNA reat, DNA danage ay, deamination activity, dsmtae activity, alkylation avy, depuration activity, xo activity, pyridine diner oning activity, integrase activity, ransposae activity, recombinase activity, polyaseatviy, igs acy, ease acy, pays ati or tyes acy. Claim St The composition of in SY, wren te enya activism ay. Chaim $32 The composton of cin wren females ey ntoduces a double stan breakin the target DNA. This endonuclease activity, which sms equ casS, i prot-yacer spec and oration dependent), ee ap. 7c (scbing experimen sling te ln ceavage avy of. thophias CCRISPRIUCas system). See aso Abstract ("We also provide in vivo evens ththe CRISPRICas system silly caves sid and teceropag oul stand DNA wikia te poospce, at spec sits” Claim $6 The composition of cai 4, wherein te DNAapting RNA isa double nol DNA argetng RNA ad th composition comprises oth rer NA and an atvalor RNA, he dpe-fomning segment of which are complementary and yb fom he soca segment ofthe DNA arin RNA, area ta cscosesompostons in which he DNAgting RNA, compra tarete-RNA (cRNA) and an acivaor RNA (ie, tracRNA), Such ompostions ar inherently present nS thermophis cel, Claim 64 A metodo sie spite motion of tt DNA, the method comping: contacting the target DNA with: Garant a. dsc etd fr ec modicaion ofa tt DDNA eg, plaid or takriophge DNA) by contacting te get DNA with DNA lrgeing RNA anda seed modifingplypeplie, Se 1p. 70 (*Overal, we have established thatthe S shermophilws CRISPRI/Cas syle ces uth backronag and psi DNA in i, Ths endoruckese act which senso eu as, pr spore pci anita dependent”) ng RNA, ora DNA poljuclvie nooing the sane, when ‘he DNALergeting RNA compris: 2 fit segment compring a uti sequen thls eomplemenary oa sequen inthe taget DNA; and) second segment that iat with site dicted mdilying polypeptide end Garneau ot al. discloses DNA-trgting RNAS. See analysis fr claim 1, above, Applicaton Nos 15'42,859 Third Paty Submision 37 CLR. § 1.290 filed Sepembe 5, 2014 Page 30 GARNEAU etal (Cite No.3) (ijase- deed mollingplyepide, ora lyre neigh sane, when th siete modifying plypeie conpi: (aan RNA- binding prion tt intact wth te DNA-argting RNA and () an city orion ht exis site-dree enzymatic avi arcu tal. dso sites modifying polypeptides, Se amis foci, ahve. Claim 65 ‘The method of claim 64, wherein the target DNA is extrachromosomal, area ta. discoses target DNA Hat setchonesoral, ach as pNT plasnid DNA, See, ep: “Squen of CRISPRL in te afemestioned 30 clones iden eet spaces ($585) al of ‘hich were logs to pNT1 sequent (Tal 1 an Fig 1)? Chaim 4 The method of cam wri ie nat activity mds axe DNA. Claim 75 Themetnd of clin 74, wherein he enzymatic city sunset, netylransrase avy, demetylseatity, DNA reir atity, DNA damage avy, deamination ati dimuaseatvity, alkylation ati, ¢epurinaion avy, oxidation atv, pyridine dnc foing atv, negra att, resposee avy, eoombinase acviy,polymerse activity, igase atv, belae avi, photolyase ato fesse actly Chain 16, Themed of cain 75, ween te DNA-modiing ena acy rucease activity. ‘Claim 77 ‘Themetnd of claim 76, wherein he ules att intodresa due stand bee inthe tet DNA, areata. dso mtd in which he enzymatic Le, doable stad ula ati modi te taget DNA, See usin faci 952. Application No: 13842859 Thi Paty Submision 37 CR, § 1.290 ied Selember 52014 Page 3] Notice of Allowance and Fee(s) Due for 14/054 414 (U.S, Patent No, 8,697,359) (Cite No. 4 This publication discloses that atthe time of the earliest priority date of the US 201410068797 family, the *797 family hhad not yet demonstrated that the CRISPR-Cas9 system could be used in eukaryotes, and it was not known whether such a system. could function in cukarotic ool, Itwas not until the third provisional patent application in this family (US 61/757, 640 fled January 28, 2013) that it was demonstrated that the CRISPR-Cas9 system could be used in eukaryotes. See The examiner's statement of reasons for allowance at page Sof Notice of Alloway. Applicaton Nos 15'42,859 Third Paty Submision 37 CLR. § 1.290 filed Sepembe 52014 Page 3? MALLetal Cite No.8) A geely modifi cell comprising econ se deed mdiing poled comprising Nal tl dls genetically nde cls comping eed rudihng polypeptide, Special, Malet ses human 2957 cel, human K5¢2 cel, and un inferences hat ae sxnealy modified to crn amanmaln expressions codpinized Ca) pron, Se aban pae' “Sie diet mdithing polypeptide” essed in chi is defied in rere [088 of US 204006879 i incude Cas). an RNA ing prion ht inerats witha DNA-arting RNA; and Malet al sds aside mollyigplypepie comping an RNA biting potion tat inka witha DNA-arsng RNA, See page 823, co. (rRNA fad oa romaly rns ened acsRNA is slice det (pron osguenoe special cave target DNA sequences matching thecRNA. an att potion tates sitet enzymatic cv, when the site of rzyatc acy is determined by the DNA-argting RNA, Nal etl dies asie-eed odin polypepide comping en acy shons site of emai aiviy is deterind by the DNA areing RNA Seepage 823col | (rRNA fsedtoeoealytransnode trarRNA isi det Cas) pron semen pel cave tant DNA squnces mating the rRNA) Claim 64 ‘Ame ose pce modication oa tres DNA the mead comprising contacting the taget DNA with Nal tal dhs methods of sie pic modifi of get DNA wing DNAargeing RNAS and seed modiyng olyepis. See abstract, (@}aDNAtngetng RNA, ora DNA polynucleotide encoding these, hein the DNA arating RNA compris 2) fist epmen comprising a rule sequence tats complementary a sequent in th tget DNA; and (h)a sco semen that inert Wiha ered modilyng polypepiean Malet al sos DNAargetng RNAS. See page 3, col 2 (Gilosng asco RNA tareRNA ison transitions (feed 00s ue RNAS (gRNAs)o dest C9 to cve sequences oie), “DNA arting RNA” sth ter suse in cm 6s defn in pags (0130) and (0136 of US 201410068797 to include “ide RNAS” hat can he @ single RNA molecule, such as @eeRNA-tarRNA fision (isle modjingplypepide, ora pyri neigh sane, hen the sieeked modilingplypepide compris (2) n RNA- binding prion tt intact with te DNAartng RNA and (an city Malet a cscs sie-dieted modifying popes. See age 23, col 2 ¢ebegun by sytesizing« human coon-pinizd version of te Cas) protein...."). Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 33 orion hl exis ste-dreed enymaticatiy “Siteiced modiyng polyepide” as used in claim 64s dling in paragraph [O18] of US 2LAO6IT to include Cas, Chaim 69 The metic 4, when ie eget DNA ipo omasoneing | Malet a divloes ste specicmniiaion of target DNA in uma 2937, el 5¢2 an ined puriiet sem els, Se abstract Claim 9 A met f promo sitespeife eavage and modification fated DNA ina el, he mad comprising iran int th cel: ale slows mets ofprmolig epic cave and ‘dtzaton of get DNA na cel itading DNA ugeting RNA and sitet ming polypeptides. Se poe 82 (DNA aring RNA or DNA poymclenide enoning he sane, herein te DNA-trgting RNA compris 2) fist segment compcinga rl equnce tats complementary a sequen in th tet DNA; and (a send erent interacts wih a ste iret modilying polypepiead Mali tal discloses DNA-‘targeting RNAs. See discussion for claim 64, above. (ijase-dreed moliingplyepide, or polyrucleide ncding te sane went sie-etd modiling polypeptide compris (0 RNA- binding portion ta nets wih he DNAting RNA; and (6) an acviy orion tht exis mule att tat crete stand breakin he ‘et DNA, Malta dco ste ined modifhng polyenes. See dicusion oe chim 8, oe vn the sith dole stand reais detent the DNA reting NA te conating ocr unde odin tat are pes or nabomologous end joining or bmolog-ireted epi, andthe get DNA. is cleaved and ined to produce adie’ DNA sun alta los otaing under condos hata pemissve ir onhonologns nd jiing or homolog dire air, esng in get DNA being cleaved andthe ona produc a modified seen, See page 24 col | an (dln geting of AVS! loin PPPIRIZC ene hick hgh mers of so logue jinn (HE) vets (cing deltos an inser wer observed upon texting wth Cos) andgRNA), ‘Chaim 93 The meld of claim, he meth further comprising ontaing he eet DN witha dnp, when th door pout, pation fe dno polynuckde,copyo te dan poyrukeide, oa prion af Nall lose method fre comping cotactng te rt DNA wi donor polyuckide.Se page 824, col. 3 songs of doar Applicaton Nos 15'42,859 Third Paty Submission 37 CER. § 1.290 filed Sepembe 5, 2014 Page 34 MALLetal Cite No.8) apy one pleut no eat DNA constuc oaitatebomologous eeuminaton (HR) meted integra), Claim 94 ‘The method of claim 92, wherein the method does at comprise contacting the cell with door polyouckte,wrein terget DNA is modi such tat ruck within th tg! DNA are dle Nal tal dls: method tt do ot use door polymcketdeuch tat ‘nucleotides within the target DNA are deleted, See page 824, col, 2 (“AS expected, NHEJ-mediated deletions for TI and T2 were centered around the re sips) Chaim 98 ‘A mend of prodcing 2 genetically modified lina sje he thd comprising (Dodge: when the DNA tating RNA compres fit segment comprising amulet sequence that is complementary toa sequence in the target DNA and poled; an the sane, wherein beste deed nodilig polypeptide comprises RNA; and (Banat prion that extiis ule avy tat casa daxble strand breakin te ret DNA wher te st fhe double strand re deterind hy the DN ging RNA the cnaing ecu under oto thar permisine ornokoologous end jn or holy repr, andthe txt DNA isla antelope 2 malied DNA segue they rung the ently mdifed cel; and (a DNA retng RNA ora DNA polrackotie cog te sn, (Wa seond eget at nrc wih ase deed odiing (ia ied mong pol ppd oapolauceodeecoing (@)an RNA Pinding pron a its with DNAagetng Mata dcloses nvoduing DNA tugting RNAS and sit-reted nila polypeptides ito 295, KS and ind pure! sr es, Seecission clin 64nd, ahve (U0) transplanting te geet nid el ino the sje. Application No: [3842859 ‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014 Page 35 MALLetal Cite No.8) Chim The method of cain, the etd uber comprising conan tcl with donor polyauclde, wherein the donor polnuceide, a prin ofthe donor pojnucie,acoy of the dnp ora patina copy ofthe donor yack integrin tet DNA, Nall dls methods fuer ompring craig the cel witha dan: polyrukide to bnegrde int the tg DNA, Session for chin 3, hove ‘Claim 1h The method of claim, wherein te metod oes at comprise conaingthe_| Natal. des method tt do ocr contacting te cl witha cell wih donor oye, wire tert DNAismodied ach tat | donor pluck sta cote with there DNA are deed ucleotides within the target DNA are deleted. ‘See discussioa for claim 54, above, Claim 102 A method of ming target DNA ina genetically modified cell hat, comprises ‘etd seuene enna an expen siete ming polypeie hemetho comping ining inthe genetically tied cella DNActgetig RNA, ora DNA polymuleotde encoding te same, vii Nal tal loss methods of moding target DNA cls ictdcing (ithe DNA targeting RNA comprises: 2) a first segment comprising a DNAargtng RNA and site-directed ming polypeptides. See discussion rucetde sequence thas complementary toa sequence inthe target DNA; | far clims 38 and 0, above, and (b) 2 sound segment tha interantswith a site ete modifying polypepie; and (ite site-directed modifying polypeptide comprises: 2) an RNAPinding gorio tt inact wit the DNA-argeng RNA and () an acy potion ‘that exhibits mules activity Dependent claims 40, 70, 95, 101, and 104 (omitted from the table) are al directed to lists of cells or organisms that comprise eukaryotic cells and organisms. As disclosed in Cite No. 4 (Notice of Allowance for U.S. Application No. 141054,414), demonstration of successful use of the CRISPR/Cas9 system in cukaryotic cells in the US 2014/0068797 family did not occur until the Application No: 13842859 ‘Thid Paty Submision 37 CER. § 1.90 filed September 52014 Page 36 filing ofthe thd provisional application (US 61/757,640; filed on January 28, 2013), which is after the January 3, 2013 online publication date of Mali et al, Mali etal. discloses use of the CRISPR/Cas9 system human 2937 cells, human K562 cells, and human induced pluripotent stem cells (see abstract, Applicaton Nos 15'42,859 Third Paty Submision 37 CER. § 1.290 filed Sepembe 5, 2014 Page 37 CONG etal (CiteNo.6) Chaim 38 A geeicaly moe el womprsing «recombinant se deed modiing poli comping an RNA binding portion that interacts witha DNA ‘an tivity pion that existe Get emai, when te sit enymatic avy is erin hy th DNA ging RNA (Congetal dso gee modtied cel comprising seed ding pole. Spey, Conta. dss nan and mse cel tata geal nied o conn mannan expression sem comprising odo ized Ca poi, Se abst an age 820 1 “Site diet ming polypeptide” essed in chi is defied in razah [188 of the 97 publon inde Cas9, Claim 64, ‘Amt of sie peri moist oes DNA the mead comping contacting tert DNA wi Conger al disses metodo st pce mieation of txt DNA wing DNA areing RNAS and sie ied modiingpolypeptibs. Se asa. {) aDNA-argtng RNA ora DNA polyno encding he sane, ‘wherein the DNA targeting RNA comprises (a first segment comprising a ruck sequence tts complemen in th tet DNA; and). seond see thinkers wih ase dete mdifing ply and (Conger al istses DNA ging RNAS. Sep. co 3 (longs ofa cleric cRNA taerRNA hd fr programabe geno eitng sig he CRISPRCs syste). “DNA argting RNA” es wed in cm 6st in paragrps[013] and [036] of US 201416797 to ine “ie RNAS at cn easing RNA molecule, such as a crRNA-traerRNA fsion, (iste ied modhng polyp, ora polyline the sung, win este malin lypeptie cmp: RNA- binding porn tht inteas wi th DNA-agtig RN ad) an ati postin hat existed nea avi. (Conger al isos sided modiing popes. Sep. 0, col. 1 (song codan-pinization of, pyogenes Cs fo exes in buman 293FT cel. “Sided maifng pope” assed in claim sdf in ere [188 of US 204006879 incu Cas. rg DNA i pole ronan ine in. Se pp. 89-20 nd ast pls, 2, 3. Cngetal slo sitespcic modification of tut chrmosonal oi in ‘oun and mouse cel that ae geet vido comprise Cad ‘Amati of prong sie pes cvag ard ofc o tegt DNA inaceh he meth comprising inducing ino thc: Conta dilses methods of promoting siv-sf cleavage and mdi fret DNA ina ely intrdaing DNA-apting RNA and Applicaton Nos 15'42,859 Third Paty Submision 37 CLR. § 1.290 filed Sepembe 52014 Page 38 CONG etal (CiteNo.6) siete moihing polypeptides. Sep, &21-47 Asenind on pg 822, Candied ology iets HDR) wa eset hE ow holy eel itu ‘pair of restriction sites, Cas9 and a mutant Cas9 nickase each catalyzed ifegaton oft epi emp into EMT loos Ascisusedon ug 82, th wildy Cad used aslo mie sit speci double stand res, which cul be pied hohe on- ‘bomologeus ead joining (NHED) including deletions o insertions) or bolo diced pi (HDR). ‘See also discussion fo aims 38 and 40, above (i/aDNAcareng RNA ora DNA poljauetide ending he ane, ‘eine DNA argetng RNA compris (2) fist segment comprising a rule sequence tats complementary a seqpece in th tget DNA; and (ba seund een ht intra witha ste iret modtying polpepie and Cong etal discloses DNAarvting RNAS. See discussion fr claim 64, above, (ives eed modyng poppe, ora plyacide noting sane, when the siete modi plypepide comps (2 RNA- binding portion tt inert wih he DNAaretig RNA; an (6) an acviy orion tt exis ucla tity tates dbl stand eck inti tet DNA, Cong etal dla steed malig plypepids. Se discus for chim 8, oe ‘here ste th double stand rea deen hy he DNA-erting RNA th coating ous under coniton tht ae pes for rorhologus en ining holo repi,and th gt DNA ‘scleved and une o ruc a modified DNA sequen, (Conger al disses contig unde condos iat ae prise for onhoologs en jing or homology direc pair, skng i get DNA bring clave anther jie i produce amid eno, Seep. £21 (Ca) wasablo mae ste-spcc duh stand reas, which ld be repaired through ether on-homolgous enjoining (NEED) inlading elo rinses) orbonolog-

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