DETAILED ACTION
Status of the Application
Claims 1-2, 5, 10, 13, 17, 19, 23, 30, 33-35, 39-40, 42, 44-45, 47-48, 50, 52, 54-55, 58, 61-62, 64, 66, 68-69, 71, 73-74, 78, 80, 86-89 are pending.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendment of claims 61, 73, 80, 86, and amendments to the specification as submitted in a communication filed on 10/24/2025 is acknowledged.
Applicant elected Group II, claims 61-62, 64, 66, 68-71, 73, 78, 80, 86, drawn in part to a system, the election of a dead Cas12 enzyme, Sau LF polymerase, and PcrA helicase, as indicated in a communication filed on 11/22/2023.
Claims 1, 2, 5, 10, 13, 17-19, 23, 30, 33-35, 39-40, 42, 44-45, 47-48, 50, 52, 54-55, 58, 64, 68, 74, 78 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention , there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 11/22/2023.
Claims 61-62, 66, 69, 71, 73, 80, 86-89 are at issue and will be examined to the extent they encompass the elected invention.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Specification
The sequence listing submitted on 8/12/2024 remains objected to for the following reasons. The Examiner has been unable to locate support for the sequence of SEQ ID NO: 26. While it is agreed that paragraph [00179] of the specification as originally filed refers to the GenBank entries WP_003870487.1, WP_034654680.1, WP_095390358.1, and WP_055343022.1, and paragraph [00186] refers to a variant of GenBank entry WP_003870487.1 having the substitutions D403A/D404A, a variant of WP_034654680.1 having the substitutions D415A/D416A, a variant of WP_095390358.1 having the substitutions D407A/D408A, and a variant of WP_055343022.1 having mutations D402A/D403A, which correspond to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 as shown in the alignments below, the specification is completely silent as to GenBank entry WP_049660019.1. There is no support for a protein having SEQ ID NO: 26 in view of the fact that there is absolutely no support in the specification as originally filed for GenBank entry WP_049660019.1. Please note that while paragraphs [00179] and [00186] of the specification as originally filed refer to a pcrA helicase from Bacillus sp. FJAT-27231 and GenBank entry WP_049660019.1 discloses the sequence of a pcrA helicase from Bacillus sp. FJAT-27231, naturally occurring variants of a protein from the same organism are a common occurrence in nature. Therefore, the mere reference to a pcrA helicase from Bacillus sp. FJAT-27231, which encompasses a genus of proteins, does not inherently provide support to the specific sequence disclosed in GenBank entry WP_049660019.1. Thus, one cannot reasonably conclude that the protein of SEQ ID NO: 26 was disclosed in the specification as originally filed. Therefore, contrary to Applicant’s assertions, there is no support for SEQ ID NO: 26 of the sequence listing. As such, the sequence listing submitted on 8/12/2024 introduces new matter into the disclosure. Applicant is required to cancel the new matter in the reply to this Office action.
Query = SEQ ID NO:25
Sbjct = DNA helicase PcrA [Thermoanaerobacter ethanolicus]; Sequence ID: WP_003870487.1
Score Expect Method Identities Positives Gaps
1438 bits(3722) 0.0 Compositional matrix adjust. 709/711(99%) 709/711(99%) 0/711(0%)
Query 1 MNNILGNLNDKQREAVMTTEGPLLILAGAGSGKTRVLTHRIAYLIKEKKVSPSNILAITF 60
MNNILGNLNDKQREAVMTTEGPLLILAGAGSGKTRVLTHRIAYLIKEKKVSPSNILAITF
Sbjct 1 MNNILGNLNDKQREAVMTTEGPLLILAGAGSGKTRVLTHRIAYLIKEKKVSPSNILAITF 60
Query 61 TNKAAEEMKTRVEDLLGYIGDLWVSTFHSACVRILRRDIDKIGYDRNFVIFDTTDQKALI 120
TNKAAEEMKTRVEDLLGYIGDLWVSTFHSACVRILRRDIDKIGYDRNFVIFDTTDQKALI
Sbjct 61 TNKAAEEMKTRVEDLLGYIGDLWVSTFHSACVRILRRDIDKIGYDRNFVIFDTTDQKALI 120
Query 121 QECLKELNLSEKQYPVKTVLNAISSAKDKMVTPEEYIYVFGNEYRSKKISEIYKLYQKKL 180
QECLKELNLSEKQYPVKTVLNAISSAKDKMVTPEEYIYVFGNEYRSKKISEIYKLYQKKL
Sbjct 121 QECLKELNLSEKQYPVKTVLNAISSAKDKMVTPEEYIYVFGNEYRSKKISEIYKLYQKKL 180
Query 181 KKNNALDFDDIIIKTIELFKESPEVLDFYQRKFKYIMVDEYQDTNMPQYHFVNMLAQKYR 240
KKNNALDFDDIIIKTIELFKESPEVLDFYQRKFKYIMVDEYQDTNMPQYHFVNMLAQKYR
Sbjct 181 KKNNALDFDDIIIKTIELFKESPEVLDFYQRKFKYIMVDEYQDTNMPQYHFVNMLAQKYR 240
Query 241 NLCVVGDDDQSIYGWRGADVKNILNFEKDYPEAKVIKLEQNYRSTKTILEAANYVIDNNI 300
NLCVVGDDDQSIYGWRGADVKNILNFEKDYPEAKVIKLEQNYRSTKTILEAANYVIDNNI
Sbjct 241 NLCVVGDDDQSIYGWRGADVKNILNFEKDYPEAKVIKLEQNYRSTKTILEAANYVIDNNI 300
Query 301 RRKKKTLWTNNEEGERIILCELENEREEAEFVIQEIINLKERENRSFRDFAILYRTNAQS 360
RRKKKTLWTNNEEGERIILCELENEREEAEFVIQEIINLKERENRSFRDFAILYRTNAQS
Sbjct 301 RRKKKTLWTNNEEGERIILCELENEREEAEFVIQEIINLKERENRSFRDFAILYRTNAQS 360
Query 361 RAFEEALMRVRIPYKVVGALRFYDRKEIKDIIAYLRILVNPYAAISFKRIINVPKRGIGA 420
RAFEEALMRVRIPYKVVGALRFYDRKEIKDIIAYLRILVNPY ISFKRIINVPKRGIGA
Sbjct 361 RAFEEALMRVRIPYKVVGALRFYDRKEIKDIIAYLRILVNPYDDISFKRIINVPKRGIGA 420
Query 421 ATIEALEATALEKDTSLFFAIDDAKVSQRAKNSLLEFKEFILELIDKKDTMTVSEVINYI 480
ATIEALEATALEKDTSLFFAIDDAKVSQRAKNSLLEFKEFILELIDKKDTMTVSEVINYI
Sbjct 421 ATIEALEATALEKDTSLFFAIDDAKVSQRAKNSLLEFKEFILELIDKKDTMTVSEVINYI 480
Query 481 LEETGYIEELEKEESEQAEGRIENLNEFLNAAYEFEESSEDKSLEAFLSGITLVSDIDLA 540
LEETGYIEELEKEESEQAEGRIENLNEFLNAAYEFEESSEDKSLEAFLSGITLVSDIDLA
Sbjct 481 LEETGYIEELEKEESEQAEGRIENLNEFLNAAYEFEESSEDKSLEAFLSGITLVSDIDLA 540
Query 541 GEIGESVVLMTLHSAKGLEFPIVFMVGMEEGIFPSFKSFTDEYELEEERRLCYVGITRSK 600
GEIGESVVLMTLHSAKGLEFPIVFMVGMEEGIFPSFKSFTDEYELEEERRLCYVGITRSK
Sbjct 541 GEIGESVVLMTLHSAKGLEFPIVFMVGMEEGIFPSFKSFTDEYELEEERRLCYVGITRSK 600
Query 601 EKLYLTYARRRNLYGKSQYSSYSRFISEIPERFLVRYHELSKPREEYRPVSSYVERKTYE 660
EKLYLTYARRRNLYGKSQYSSYSRFISEIPERFLVRYHELSKPREEYRPVSSYVERKTYE
Sbjct 601 EKLYLTYARRRNLYGKSQYSSYSRFISEIPERFLVRYHELSKPREEYRPVSSYVERKTYE 660
Query 661 KAQYNLGDKVEHKIWGIGTVVKVEGEEITVAFPNVGIKKLDLKFAPIKAIS 711
KAQYNLGDKVEHKIWGIGTVVKVEGEEITVAFPNVGIKKLDLKFAPIKAIS
Sbjct 661 KAQYNLGDKVEHKIWGIGTVVKVEGEEITVAFPNVGIKKLDLKFAPIKAIS 711
Query = SEQ ID NO:27
Sbjct = DNA helicase PcrA [Priestia]; Sequence ID: WP_034654680.1
NW Score Identities Positives Gaps
3756 747/749(99%) 747/749(99%) 0/749(0%)
Query 1 MFDIGGENVNFLSEKLLTGLNPQQQEAVKTTDGPLLLMAGAGSGKTRVLTHRIAFLMAEK 60
MFDIGGENVNFLSEKLLTGLNPQQQEAVKTTDGPLLLMAGAGSGKTRVLTHRIAFLMAEK
Sbjct 1 MFDIGGENVNFLSEKLLTGLNPQQQEAVKTTDGPLLLMAGAGSGKTRVLTHRIAFLMAEK 60
Query 61 EVAPWNILAITFTNKAAREMRERVSNIVGGVAEDIWISTFHSMCVRILRRDIDRIGFNRN 120
EVAPWNILAITFTNKAAREMRERVSNIVGGVAEDIWISTFHSMCVRILRRDIDRIGFNRN
Sbjct 61 EVAPWNILAITFTNKAAREMRERVSNIVGGVAEDIWISTFHSMCVRILRRDIDRIGFNRN 120
Query 121 FTILDSTDQLSVIKNILKDQNIDPKKFDPRSLLGSISSAKNELKVAEEFDKTATGPYEEV 180
FTILDSTDQLSVIKNILKDQNIDPKKFDPRSLLGSISSAKNELKVAEEFDKTATGPYEEV
Sbjct 121 FTILDSTDQLSVIKNILKDQNIDPKKFDPRSLLGSISSAKNELKVAEEFDKTATGPYEEV 180
Query 181 VSKVYKEYEKRLKKNQALDFDDLIMTTIQLFKRVPEVLTYYQRKFQYIHVDEYQDTNHAQ 240
VSKVYKEYEKRLKKNQALDFDDLIMTTIQLFKRVPEVLTYYQRKFQYIHVDEYQDTNHAQ
Sbjct 181 VSKVYKEYEKRLKKNQALDFDDLIMTTIQLFKRVPEVLTYYQRKFQYIHVDEYQDTNHAQ 240
Query 241 YMLVRLLAARFENVCVVGDSDQSIYRWRGADITNILSFEKDYPKAKTILLEQNYRSTKTI 300
YMLVRLLAARFENVCVVGDSDQSIYRWRGADITNILSFEKDYPKAKTILLEQNYRSTKTI
Sbjct 241 YMLVRLLAARFENVCVVGDSDQSIYRWRGADITNILSFEKDYPKAKTILLEQNYRSTKTI 300
Query 301 LAAANGVIANNMNRKVKNLWTENDEGQKIYHYQAMSEHDEAQFVARKIKEAVDSGKRKYS 360
LAAANGVIANNMNRKVKNLWTENDEGQKIYHYQAMSEHDEAQFVARKIKEAVDSGKRKYS
Sbjct 301 LAAANGVIANNMNRKVKNLWTENDEGQKIYHYQAMSEHDEAQFVARKIKEAVDSGKRKYS 360
Query 361 DFAILYRTNAQSRVMEEVLLKSNINYTIVGGIKFYDRKEIKDLLAYLRLIANQDAAISLA 420
DFAILYRTNAQSRVMEEVLLKSNINYTIVGGIKFYDRKEIKDLLAYLRLIANQD ISLA
Sbjct 361 DFAILYRTNAQSRVMEEVLLKSNINYTIVGGIKFYDRKEIKDLLAYLRLIANQDDDISLA 420
Query 421 RIVNVPKRGVGATSVDKVANYGNVHDISIFKALDEVELMGLTGKATKALRDFQSMISNLA 480
RIVNVPKRGVGATSVDKVANYGNVHDISIFKALDEVELMGLTGKATKALRDFQSMISNLA
Sbjct 421 RIVNVPKRGVGATSVDKVANYGNVHDISIFKALDEVELMGLTGKATKALRDFQSMISNLA 480
Query 481 QMQDYMSVTELVEQVLERTGYREALKVEKTIEAQSRLENIDEFLSVTKTFEEASEDKSLV 540
QMQDYMSVTELVEQVLERTGYREALKVEKTIEAQSRLENIDEFLSVTKTFEEASEDKSLV
Sbjct 481 QMQDYMSVTELVEQVLERTGYREALKVEKTIEAQSRLENIDEFLSVTKTFEEASEDKSLV 540
Query 541 AFLTDLALVADIDKLEDEEEEENEQVILMTLHSAKGLEFPVVFLMGMEEGVFPHSRSLFE 600
AFLTDLALVADIDKLEDEEEEENEQVILMTLHSAKGLEFPVVFLMGMEEGVFPHSRSLFE
Sbjct 541 AFLTDLALVADIDKLEDEEEEENEQVILMTLHSAKGLEFPVVFLMGMEEGVFPHSRSLFE 600
Query 601 DNEMEEERRLAYVGITRAEQELYLLNAQMRTLFGKTNVNPKSRFIGEIPSELVESLNEAM 660
DNEMEEERRLAYVGITRAEQELYLLNAQMRTLFGKTNVNPKSRFIGEIPSELVESLNEAM
Sbjct 601 DNEMEEERRLAYVGITRAEQELYLLNAQMRTLFGKTNVNPKSRFIGEIPSELVESLNEAM 660
Query 661 RKPAAGRSGASASPFAARRQAAVAKTKLVSTGSESIGWSVGDKAEHKKWGIGTVVSVKGE 720
RKPAAGRSGASASPFAARRQAAVAKTKLVSTGSESIGWSVGDKAEHKKWGIGTVVSVKGE
Sbjct 661 RKPAAGRSGASASPFAARRQAAVAKTKLVSTGSESIGWSVGDKAEHKKWGIGTVVSVKGE 720
Query 721 GDSKELDIAFPSPTGVKRLLAKFAPVTKV 749
GDSKELDIAFPSPTGVKRLLAKFAPVTKV
Sbjct 721 GDSKELDIAFPSPTGVKRLLAKFAPVTKV 749
Query = SEQ ID NO:28
Sbjct = DNA helicase PcrA [Peribacillus]; Sequence ID: WP_095390358.1
Score Expect Method Identities Positives Gaps
1536 bits(3977) 0.0 Compositional matrix adjust. 746/748(99%) 746/748(99%) 0/748(0%)
Query 1 MQYLAEKLLIGLNEQQQKAVKATDGPLLIMAGAGSGKTRVLTHRIAYLMVEKEVAPWNIL 60
MQYLAEKLLIGLNEQQQKAVKATDGPLLIMAGAGSGKTRVLTHRIAYLMVEKEVAPWNIL
Sbjct 1 MQYLAEKLLIGLNEQQQKAVKATDGPLLIMAGAGSGKTRVLTHRIAYLMVEKEVAPWNIL 60
Query 61 AITFTNKAAREMKERIRAILGGASEDIWISTFHSMCVRILRRDIDRIGFNRNFSILDTTD 120
AITFTNKAAREMKERIRAILGGASEDIWISTFHSMCVRILRRDIDRIGFNRNFSILDTTD
Sbjct 61 AITFTNKAAREMKERIRAILGGASEDIWISTFHSMCVRILRRDIDRIGFNRNFSILDTTD 120
Query 121 QQSVIKQIMKDRNMDTKKYDYRAILGSISSAKNELVGPEEYLKTASDYFTKVTADVYTEY 180
QQSVIKQIMKDRNMDTKKYDYRAILGSISSAKNELVGPEEYLKTASDYFTKVTADVYTEY
Sbjct 121 QQSVIKQIMKDRNMDTKKYDYRAILGSISSAKNELVGPEEYLKTASDYFTKVTADVYTEY 180
Query 181 QKRLRKNSALDFDDLIMMTIQLFQLVPEVLEYYQRKFQYIHVDEYQDTNRAQYMLVKLLA 240
QKRLRKNSALDFDDLIMMTIQLFQLVPEVLEYYQRKFQYIHVDEYQDTNRAQYMLVKLLA
Sbjct 181 QKRLRKNSALDFDDLIMMTIQLFQLVPEVLEYYQRKFQYIHVDEYQDTNRAQYMLVKLLA 240
Query 241 SRFRNLCVVGDSDQSIYRWRGADIANILSFEKDYPNANMIFLEQNYRSTKKILAAANKVI 300
SRFRNLCVVGDSDQSIYRWRGADIANILSFEKDYPNANMIFLEQNYRSTKKILAAANKVI
Sbjct 241 SRFRNLCVVGDSDQSIYRWRGADIANILSFEKDYPNANMIFLEQNYRSTKKILAAANKVI 300
Query 301 DNNQNRKPKNLWTENADGNKIFYYRADNEQGEAQFVAGKINELVQDGSRKYSDIAILYRT 360
DNNQNRKPKNLWTENADGNKIFYYRADNEQGEAQFVAGKINELVQDGSRKYSDIAILYRT
Sbjct 301 DNNQNRKPKNLWTENADGNKIFYYRADNEQGEAQFVAGKINELVQDGSRKYSDIAILYRT 360
Query 361 NAQSRVMEEVLLKSNINYAIVGGTKFYDRKEIKDLLAYLRLIANPDAAISLRRVINVPKR 420
NAQSRVMEEVLLKSNINYAIVGGTKFYDRKEIKDLLAYLRLIANPD ISLRRVINVPKR
Sbjct 361 NAQSRVMEEVLLKSNINYAIVGGTKFYDRKEIKDLLAYLRLIANPDDDISLRRVINVPKR 420
Query 421 GIGATSMDKVADYADQYDVSIYKALESVEMAGISGKATKAAREFHTLITNYTNQQEYLSV 480
GIGATSMDKVADYADQYDVSIYKALESVEMAGISGKATKAAREFHTLITNYTNQQEYLSV
Sbjct 421 GIGATSMDKVADYADQYDVSIYKALESVEMAGISGKATKAAREFHTLITNYTNQQEYLSV 480
Query 481 TELVEEVIEKTGYREMLQAEKTIESQSRLENIDEFLSVTKAFEDNSEDKSLVGFLTDLAL 540
TELVEEVIEKTGYREMLQAEKTIESQSRLENIDEFLSVTKAFEDNSEDKSLVGFLTDLAL
Sbjct 481 TELVEEVIEKTGYREMLQAEKTIESQSRLENIDEFLSVTKAFEDNSEDKSLVGFLTDLAL 540
Query 541 VADIDQLDENTEEATNTVTLMTLHSAKGLEYPVVFLLGLEEGVFPHSRSLMDEEEMEEER 600
VADIDQLDENTEEATNTVTLMTLHSAKGLEYPVVFLLGLEEGVFPHSRSLMDEEEMEEER
Sbjct 541 VADIDQLDENTEEATNTVTLMTLHSAKGLEYPVVFLLGLEEGVFPHSRSLMDEEEMEEER 600
Query 601 RLAYVGITRAENELFISNAQMRTLYGRTSMNPVSRFISEIPEELLEDLKPKPAPRARQTP 660
RLAYVGITRAENELFISNAQMRTLYGRTSMNPVSRFISEIPEELLEDLKPKPAPRARQTP
Sbjct 601 RLAYVGITRAENELFISNAQMRTLYGRTSMNPVSRFISEIPEELLEDLKPKPAPRARQTP 660
Query 661 FSSSRTGSPSTASTRKVPAFGKAVSAPSATGGEEIGWAVGDRASHKKWGIGTVVSVKGEG 720
FSSSRTGSPSTASTRKVPAFGKAVSAPSATGGEEIGWAVGDRASHKKWGIGTVVSVKGEG
Sbjct 661 FSSSRTGSPSTASTRKVPAFGKAVSAPSATGGEEIGWAVGDRASHKKWGIGTVVSVKGEG 720
Query 721 EGKELDIAFPSPIGIKRLLAKFAPVEKA 748
EGKELDIAFPSPIGIKRLLAKFAPVEKA
Sbjct 721 EGKELDIAFPSPIGIKRLLAKFAPVEKA 748
Query = SEQ ID NO:29
Sbjct = DNA helicase PcrA [Paraclostridium sordellii], Sequence ID: WP_055343022.1
Score Expect Method Identities Positives Gaps
1509 bits(3906) 0.0 Compositional matrix adjust. 738/740(99%) 738/740(99%) 0/740(0%)
Query 1 MDIDSLNPAQKEAVLKTEGPVLILAGAGSGKTRVLTTRIAHLMKNKGVHPSNILAITFTN 60
MDIDSLNPAQKEAVLKTEGPVLILAGAGSGKTRVLTTRIAHLMKNKGVHPSNILAITFTN
Sbjct 1 MDIDSLNPAQKEAVLKTEGPVLILAGAGSGKTRVLTTRIAHLMKNKGVHPSNILAITFTN 60
Query 61 KAANEMKERVEETIDSDVKDMWITTFHSCCVRMLRKDINRIGYNRSFVIYDSSDQVTLVK 120
KAANEMKERVEETIDSDVKDMWITTFHSCCVRMLRKDINRIGYNRSFVIYDSSDQVTLVK
Sbjct 61 KAANEMKERVEETIDSDVKDMWITTFHSCCVRMLRKDINRIGYNRSFVIYDSSDQVTLVK 120
Query 121 DCLKELNLSDKTFEPKVVISYISGAKDKLLTPEQYKDMHRSDARMSKIAAVYTLYQDRLK 180
DCLKELNLSDKTFEPKVVISYISGAKDKLLTPEQYKDMHRSDARMSKIAAVYTLYQDRLK
Sbjct 121 DCLKELNLSDKTFEPKVVISYISGAKDKLLTPEQYKDMHRSDARMSKIAAVYTLYQDRLK 180
Query 181 RNSALDFDDLIIKTVELLKKEESVLAYYRNKFKYIMVDEYQDTSKAQYEFIKLLAKEHQN 240
RNSALDFDDLIIKTVELLKKEESVLAYYRNKFKYIMVDEYQDTSKAQYEFIKLLAKEHQN
Sbjct 181 RNSALDFDDLIIKTVELLKKEESVLAYYRNKFKYIMVDEYQDTSKAQYEFIKLLAKEHQN 240
Query 241 ICVVGDDDQSIYGWRGADIRNILEFERDYSDVHVVKLEQNYRSTQVILDAANTVISNNVE 300
ICVVGDDDQSIYGWRGADIRNILEFERDYSDVHVVKLEQNYRSTQVILDAANTVISNNVE
Sbjct 241 ICVVGDDDQSIYGWRGADIRNILEFERDYSDVHVVKLEQNYRSTQVILDAANTVISNNVE 300
Query 301 RKRKKLWSEQKEGEKIKIQVNADEIEEADFVADSIWKIHEKENKPFKDFAVLYRANAQAR 360
RKRKKLWSEQKEGEKIKIQVNADEIEEADFVADSIWKIHEKENKPFKDFAVLYRANAQAR
Sbjct 301 RKRKKLWSEQKEGEKIKIQVNADEIEEADFVADSIWKIHEKENKPFKDFAVLYRANAQAR 360
Query 361 AIEDALNRSQIPYNIYGGTKFYERKEIKDLVAYLRVLQNVQAAISLKRIINVPKRGIGLR 420
AIEDALNRSQIPYNIYGGTKFYERKEIKDLVAYLRVLQNVQ ISLKRIINVPKRGIGLR
Sbjct 361 AIEDALNRSQIPYNIYGGTKFYERKEIKDLVAYLRVLQNVQDDISLKRIINVPKRGIGLR 420
Query 421 TIEKIEDRASLKQESIFSVLLDVDTNSDISTKARANINGFVDLIGTLRVIKDAYSVSKLI 480
TIEKIEDRASLKQESIFSVLLDVDTNSDISTKARANINGFVDLIGTLRVIKDAYSVSKLI
Sbjct 421 TIEKIEDRASLKQESIFSVLLDVDTNSDISTKARANINGFVDLIGTLRVIKDAYSVSKLI 480
Query 481 ERVLDVTGYLDELEKDKSEESQDRIDNLKEFISIAIEFENSSEEKDLETFLTNVALTSSE 540
ERVLDVTGYLDELEKDKSEESQDRIDNLKEFISIAIEFENSSEEKDLETFLTNVALTSSE
Sbjct 481 ERVLDVTGYLDELEKDKSEESQDRIDNLKEFISIAIEFENSSEEKDLETFLTNVALTSSE 540
Query 541 SGEEEDDRVSLMTIHTSKGLEFPVVFLIGMEEGLFPISRAVRSMSESDIEEERRLCYVGI 600
SGEEEDDRVSLMTIHTSKGLEFPVVFLIGMEEGLFPISRAVRSMSESDIEEERRLCYVGI
Sbjct 541 SGEEEDDRVSLMTIHTSKGLEFPVVFLIGMEEGLFPISRAVRSMSESDIEEERRLCYVGI 600
Query 601 TRAKKELYMTLTKKRTLYGKTNPSIQSRFMEELPQECIEKLNEEVHELSYSKANYNILDK 660
TRAKKELYMTLTKKRTLYGKTNPSIQSRFMEELPQECIEKLNEEVHELSYSKANYNILDK
Sbjct 601 TRAKKELYMTLTKKRTLYGKTNPSIQSRFMEELPQECIEKLNEEVHELSYSKANYNILDK 660
Query 661 YKQKYMKSMNKASVATKVNATIKESKPESNVDDIKLGSKVHHPKFGVGTVVSASGSDLTI 720
YKQKYMKSMNKASVATKVNATIKESKPESNVDDIKLGSKVHHPKFGVGTVVSASGSDLTI
Sbjct 661 YKQKYMKSMNKASVATKVNATIKESKPESNVDDIKLGSKVHHPKFGVGTVVSASGSDLTI 720
Query 721 AFNNQGIKKINKEYTTLDLL 740
AFNNQGIKKINKEYTTLDLL
Sbjct 721 AFNNQGIKKINKEYTTLDLL 740
The amendment filed 10/24/2025 is objected to under 35 U.S.C. 132 (a) because it introduces new matter into the disclosure. 35 U.S.C. 132(a) states that no amendment shall introduce new matter into the disclosure of the invention. The added material which is not supported by the original disclosure is as follows. Paragraphs [0179] and [00186] of the amended specification refer to the GenBank entry WP_049660019.1 and a protein that comprises SEQ ID NO: 26 which is a variant of the protein of GenBank entry WP_049660019.1. However, as indicated above, the specification as originally filed makes no mention whatsoever of GenBank entry WP_049660019.1, let alone a variant of the protein of GenBank entry WP_049660019.1. having mutations D407A/D408A. Applicant is required to cancel the new matter in the reply to this Office action.
Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA )
Claims 73, 86 and 89 were rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention. In view of Applicant’s amendment of claim 73 and 61, this rejection is hereby withdrawn.
Claim Rejections - 35 USC § 112(a) or First Paragraph (pre-AIA )
Claim 86 remains rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention. This rejection is necessitated by the introduction of new matter.
As set forth in MPEP 2163 (I)(B), new or amended claims which introduce elements or limitations that are not supported by the as-filed disclosure violate the written description requirement. See, e.g., In re Lukach, 442 F.2d 967, 169 USPQ 795 (CCPA 1971) (subgenus range was not supported by generic disclosure and specific example within the subgenus range); In re Smith, 458 F.2d 1389, 1395, 173 USPQ 679, 683 (CCPA 1972) (an adequate description of a genus may not support claims to a subgenus or species within the genus).
Claim 86 requires the polypeptide of SEQ ID NO: 26. The Examiner is unable to locate adequate support in the specification for the polypeptide of SEQ ID NO: 26. As explained above, there is absolutely no support in the specification as originally filed for GenBank entry WP_049660019.1, let alone for a variant of the protein of GenBank entry WP_049660019.1 having the D407A/D408A substitutions. Thus there is no indication that a system that requires the polypeptide of SEQ ID NO: 26 was within the scope of the invention as conceived by Applicant at the time of the invention. Accordingly, Applicant is required to cancel the new matter in the response to this Office Action.
Claim Rejections - 35 USC § 103 (AIA )
Claims 73 and 89 are rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), and further in view of Deiman et al. (Molecular Biotechnology 20:163-179, 2002). This rejection is necessitated by amendment of claim 73.
Mandell teaches that binding of a guide RNA to a region of a target double-stranded nucleic acid disrupts the interaction between the two strands of the target nucleic acid and thereby creates a loop structure (R-loop) that exposes the non-complementary strand to the guide RNA, such that this exposed strand can be subjected to hybridization with primer for extension by an appropriate polymerase (pages 17-18, paragraph [0060]). Mandell also teaches that the loop structure created by CRISPR-Cas systems can be accessible to other enzymes and that this loop structure can be further utilized as a template to initiate primer hybridization and interact with polymerases for amplification (page 18, lines 3-6). Mandell teaches a system for amplifying a target double-stranded nucleic acid, wherein said system comprises CRISPR-Cas systems, primers and a polymerase (pages 17-18, paragraphs [0060]-[0061]), wherein the system comprises (a) a first guide RNA (crRNA) that contains a region complementary to a region of a first strand of the target double-stranded nucleic acid and a first CRISPR-Cas protein, (b) a second guide RNA that contains a region complementary to a region of a second strand of the target double-stranded nucleic acid, and a second CRISPR-Cas protein, (c) a pair of primers, wherein the first primer is complementary to a region of the second strand of the target double-stranded nucleic acid, and the second primer is complementary to a region of the first strand of the target double-stranded nucleic acid, and a polymerase (pages 19-20, paragraphs [0066]-[0068]). Mandell teaches that the polymerase of his system can be a strand-displacing polymerase, such as Bst DNA polymerase, DNA polymerase I Klenow fragment, phi29 DNA polymerase, or Taq polymerase (page 26, paragraph [0087]). Mandell teaches that the amplification of the desired nucleic acid can be carried out under isothermal conditions (i.e., same temperature) and that the amplification reaction temperature can be at 37 °C (page 27, paragraph [0088]), thus teaching that the polymerase and CRISRP-Cas enzyme are active at 37 °C. Mandell teaches that his system includes detection labels to detect the amplified products (page 26, paragraph [0086]), such as biotin, radioactive or fluorescent labels (page 28, paragraphs [0092]-[0093]), dyes (page 27, paragraph [0091]), and molecular beacons (page 28, paragraph [0094]). Mandell teaches that the CRISPR-Cas protein is a Cas9 protein which is enzymatically inactive (dead Cas9; page 6, paragraph [0028]; page 24, paragraph [0080]). Mandell does not teach the use of a helicase in their system.
Kong et al. teach helicase dependent amplification of nucleic acids, wherein said amplification is carried out isothermally in the presence of a DNA polymerase (Abstract). Kong et al. teach several helicases that can be used in their method including helicases from E. coli, which is a mesophile (grows at 37 °C) and a PcrA helicase (paragraph [0015]). Kong et al. teach that the helicase preparation for the amplification contains single strand binding proteins such as T4 gene32 single strand binding protein (paragraphs [0017], [0106]) and cofactors such as ATP, including a concentration of 3 mM (paragraph [0110]). Kong et al. teach that the amplification can be carried out isothermally at a temperature between 20-75 °C (paragraph [0011]), including 37 °C (Example IX, paragraph [0259]). Kong et al. teach that increasing the temperature assists in initial unwinding of the target nucleic acid by the helicase and that the amplification by the polymerase can proceed at a single temperature (paragraph [0125]). Korfhage teaches that isothermal amplification of nucleic acids do not require a thermocycler but that isothermal amplification methods are error-prone, specifically unspecific amplification due to mispairing of primers (paragraph [0007]). Korfhage teaches that to avoid the generation of these side products, the reaction components are heated separately and mixed at higher temperatures so that the primers can anneal to their intended target (paragraph [0007]). Neither Kong et al. nor Korfhage teach a CRISPR amplification system.
Deiman et al. teach that nucleic acid sequence based amplification (NASBA) requires a primer having a T7 promoter sequence at the 5’-end of the primer (upstream from the region that is complementary with the target nucleic acid; page 165, left column DNA NASBA; page 167, right column, Primer and Probe Design).
Claims 73 and 79 as amended are directed in part to a system for helicase-dependent amplification of a target double stranded nucleic acid, wherein said system comprises two amplification CRISPR/Cas complexes, a pair of primers, a polymerase, a PcrA helicase, a T4 gene 32 single-stranded binding protein, ATP at a concentration of about 0.75 mM-5 mM, and a detection system for detecting amplification of the target double stranded nucleic acid, wherein each of the CRISPR/Cas complexes can comprise a dead Cas9 protein, or a Cas9 protein, and a guide molecule, wherein one of guide molecules guides one of the CRISPR/Cas complexes to the first strand of the target nucleic acid, and the other guide molecule guides the other CRISPR/Cas complex to the second strand of the target nucleic acid, wherein one of the primers in the pair is complementary to the first strand of the target nucleic acid and comprises the T7 RNA polymerase promoter upstream of the portion of the primer that is complementary to the first strand of the target nucleic acid, and the other primer is complementary to the second strand of the target nucleic acid, wherein the polymerase is a full length Bst DNA polymerase, a phi29 DNA polymerase, a Taq polymerase, or a DNA polymerase I Klenow fragment, and wherein the CRISPR Cas9 protein, the polymerase and the helicase can be enzymatically active at about 37 °C.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the system of Martell with the system of Kong et al. and use a primer that has a T7 RNA polymerase promoter upstream from the region of the primer that is complementary to one of the strands. A person of ordinary skill in the art is motivated to add a helicase in the system of Martell for the benefit of adding an enzyme that would allow further unwinding of the target double stranded nucleic acid so that primers could bind to the desired targets and amplification could take place via the polymerase. A person of ordinary skill in the art is motivated to use a primer having a T7 RNA polymerase promoter upstream from the region of the primer that is complementary to one of the strands of the target nucleic acid because primers that have a T7 RNA polymerase promoter at the 5” region are required for nucleic acid sequence based amplification as evidenced by Deiman et al. A person of ordinary skill in the art is motivated to include the CRISPR amplification components of Mandell in the helicase-dependent amplification system of Kong et al. because the art as evidenced by Korfhage teaches that isothermal amplification methods, such as a helicase-dependent amplification method, are error-prone unless a denaturing step (e.g., heating to separate the strands) is used to allow initial unwinding of the target nucleic acid and binding of the primers to their intended target. As disclosed by Mandell, the loop structure created by CRISPR-Cas systems can be accessible to other enzymes and that this loop structure can be further utilized as a template to initiate primer hybridization and interact with polymerases for amplification. The CRISPR-Cas system could allow the replacement of the denaturing step (initial unwinding) as it provides a loop for the helicase to act on the target nucleic acid. One of ordinary skill in the art has a reasonable expectation of success at combining the amplification systems of Mandell and Kong et al. because all that is required is the inclusion of the helicase compositions of Kong et al. in the system of Mandell et al. or the inclusion of the CRISPR/Cas complexes of Mandell into the system of Kong et al. Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 61, 62, 66, 69, 80, 87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS) and Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS).
Claims 62 and 80 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), and further in view of Begemann et al. (U.S. Publication No. 2017/0233756 published 8/17/2017).
Claims 71 and 88 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), and further in view of Gootenberg et al. (Science 360:439-444, published online 2/15/2018; cited in the IDS).
These rejections have been discussed at length in the prior Office action. They are maintained for the reasons of record and those set forth below.
Applicant argues that there is no motivation to modify the system of Kong et al. because they already solve the alleged “error prone” problem through its established thermal initiation approach and that the Examiner provides no explanation as to why one of skill in the art would abandon the system of Kong et al. for a significantly more complex CRISPR integration. Applicant states that Kong et al. do not teach that CRISPR-generated R loops could serve as enhanced initiation sites for helicase dependent amplification. Applicant states that the Examiner’s interpretation that the E. coli UvrD helicase of Kong et al. makes the PcrA variants of SEQ ID NO: 25-29 obvious is fundamentally flawed. Applicant states that the prior art provides no guidance that these particular conserved positions should be targeted for mutation and that alanine substitutions at these specific sites would enhance performance in CRISPR-integrated systems, or that these particular mutations would provide superior function compared to wild-type helicases. Applicant states that Mandell’s R-loops are designed for thermal cycling amplification with conventional PCR, where thermal cycling provides all necessary DNA unwinding and that Kong et al. teach helicases that are optimized for heating-initiated unwinding in isothermal conditions. Applicant states that neither reference discloses the technical insight that combining these independently functional systems could yield synergistic benefits, or provide guidance for the complex coordination required between CRISPR R-loop generation and helicase-mediated unwinding. Applicant states that Mandell provides no teaching that CRISPR-generated R-loops could address these limitations. Applicant states that the system of the invention provides unexpected synergistic advantages, namely eliminating heating requirements through CRISPR-initiated unwinding while providing enhanced substrate accessibility, at a temperature of about 37 °C. Applicant states that the unexpected coordination between CRISPR R-loop generation and helicase activity provides performance advantages that result from the novel technical insight that CRISPR R-loops can serve as enhanced initiation sites for helicase activity.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejections. The Examiner acknowledges the teachings of Kong et al. and Mandell but disagrees with Applicant’s contention that the claimed invention is not obvious over the prior art.
With regard to the argument that there is no motivation to modify the system of Kong et al. because they already solve the alleged “error prone” problem through its established thermal initiation approach, as previously indicated, Kong et al. teach that increasing the temperature is needed in initial unwinding of the target nucleic acid and Korfhage teaches that isothermal amplification methods have can be error prone due to mispairing of primers. The use of a helicase in the amplification method of Kong et al. simply allows unwinding of DNA under isothermal conditions but does not have an effect on the mispairing of primers. The use of CRISPR in the method of Kong et al. would avoid having to increase the temperature for initial unwinding because the CRISPR system of Mandell would provide the R-loop, which is obtained as a result of initial unwinding of the target nucleic acid. Moreover, one of skill in the art would expect that the use of the CRISPR system of Mandell would increase specificity of primer binding because the guide RNAs would hybridize to the target nucleic acid and the only area exposed for initial primer binding is the R-loop. Therefore, contrary to Applicant’s assertions, one of skill in the art would be highly motivated to add the CRISPR system of Mandell to the method of Kong et al.
With regard to the argument that Kong et al. do not teach that CRISPR-generated R loops could serve as enhanced initiation sites for helicase dependent amplification, it is noted that as set forth in MPEP § 2143(G), the courts have made clear that the teaching, suggestion, or motivation test is flexible and an explicit suggestion to combine the prior art is not necessary. The motivation to combine may be implicit and may be found in the knowledge of one of ordinary skill in the art, or, in some cases, from the nature of the problem to be solved. In the instant case, it was well known in the art and taught by Kong et al., that (i) helicases unwind DNA and that amplification with a polymerase requires unwinding DNA for the polymerase to amplify the target nucleic acid, and (ii) binding of a guide RNA to a region of a target double stranded nucleic acid disrupts the interaction of the two strands and creates a loop that exposes the non-complementary strand to the guide RNA, thus unwinding the double stranded nucleic acid around the region of the target nucleic acid where the guide RNA binds, as taught by Mandell. Therefore, while it is agreed that Kong et al. do not refer to CRISPR-generated R loops or their potential use as initiation sites for helicase dependent amplification, one of skill in the art based on the teachings of Mandell and Kong et al. could reasonably conclude that R-loops are the result of unwinding a double stranded nucleic acid and that they can be used as an initiation site for helicase amplification.
With regard to the argument that the Examiner’s interpretation that the E. coli UvrD helicase of Kong et al. makes the PcrA variants of SEQ ID NO: 25-29 obvious is fundamentally flawed, it is noted that the Examiner has not made the argument that the E. coli UvrD helicase of Kong et al. makes the PcrA variants of SEQ ID NO: 25-29 obvious. Moreover, arguments regarding the polypeptides of SEQ ID NO: 25-29 are irrelevant to the instant discussion because claim 87, which requires the proteins of SEQ ID NO: 25-26, is not included in any of the rejections set forth above.
With regard to the argument that Mandell’s R-loops are designed for thermal cycling amplification with conventional PCR, where thermal cycling provides all necessary DNA unwinding and that Kong et al. teach helicases that are optimized for heating-initiated unwinding in isothermal conditions, it is reiterated herein that Mandell teaches that their amplification method can be carried out under isothermal conditions and that the amplification reaction temperature can be at 37 °C (page 27, paragraph [0088]).
With regard to the argument that neither reference discloses the technical insight that combining these independently functional systems could yield synergistic benefits, or provide guidance for the complex coordination required between CRISPR R-loop generation and helicase-mediated unwinding, it is noted that one of skill in the art did not need to know a priori of synergistic effects to be motivated to combine the teachings of Mandell and Kong et al. and to reasonably expect that the method of Mandell and Kong et al. would provide an advantage. It is reiterated herein that a person of ordinary skill in the art is motivated to add a helicase in the system of Martell for the benefit of adding an enzyme that would allow further unwinding of the target double stranded nucleic acid so that primers could bind to the desired targets and amplification could take place via the polymerase. As explained above, a person of ordinary skill in the art is motivated to add the CRISPR system of Mandell to the method of Kong et al. to (i) avoid having to increase the temperature for initial unwinding because the CRISPR system of Mandell would provide the initial unwinding via the R-loop, which is obtained as a result of unwinding of the target nucleic acid, and (ii) to increase specificity of primer binding because the guide RNAs would hybridize to the target nucleic acid and the only area exposed for initial primer binding is the R-loop, which is located at the target nucleic.
With regard to the argument that the system of the invention provides unexpected synergistic advantages, namely eliminating heating requirements through CRISPR-initiated unwinding while providing enhanced substrate accessibility, at a temperature of about 37 °C, it is noted that Mandell teaches that their method can be carried out at about 37 C and the R-loop formation, which is the result of double stranded nucleic acid unwinding, does not require heating. Therefore, it is not believed that substrate accessibility at a temperature of about 37 °C is unexpected. While Applicant argues that
the unexpected coordination between CRISPR R-loop generation and helicase activity provides performance advantages that result from the novel technical insight that CRISPR R-loops can serve as enhanced initiation sites for helicase activity, it is noted that Mandell specifically teaches that the R-loop can be accessible to other enzymes and that this loop structure can be further utilized as a template to initiate primer hybridization and interact with polymerases for amplification (page 18, lines 3-6). The use of a helicase in the method of Mandell et al simply allows further unwinding of the target nucleic acid. Therefore, there is nothing unexpected about unwinding DNA with a helicase once initial unwinding is present. Please note that an R-loop is a region of unwound DNA. Thus, for the reasons of record and those set forth above, one cannot reasonably conclude that the claimed invention is not obvious over the prior art of record.
Claims 61, 62, 66, 69, 80, 86-87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1 6/15/2010), and GenBank accession No. WP_003870487.1 (5/7/2013).
Claims 61, 62, 66, 69, 80, 86-87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1), and GenBank accession No. WP_049660019.1 (7/24/2015).
Claims 61, 62, 66, 69, 80, 86-87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1), and GenBank accession No. WP_034654680.1 (12/23/2014).
Claims 61, 62, 66, 69, 80, 86-87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1), and GenBank accession No. WP_095390358.1 (8/30/2017).
Claims 61, 62, 66, 69, 80, 86-87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1), and GenBank accession No. WP_055343022.1 (10/27/2015).
These reelections have been discussed at length in the prior Office action. They are maintained for the reasons of record and those set forth above.
Applicant argues that the teachings of Ma et al. in isolation cannot render obvious the claimed PcrA variants with mutations at different positions from different organisms in a fundamentally different application context. Applicant states that Ma et al. did not examine UvrD mutations for integration with CRISPR systems or isothermal amplification applications. Applicant states that even where GenBank accessions contain sequences that differ by only two amino acids from the claimed sequences, the obviousness of these specific mutations cannot be established without hindsight bias. Applicant states that the GenBank entries cited disclose wild-type PcrA sequences without the specific substitutions claimed. Applicant states that the claimed sequences represent engineered variants with strategic mutations specifically for CRISPR-integrated systems. Applicant states that the rejections assume obviousness without adequate motivation for the particular motivations. According to Applicant, the prior art does not provide guidance that these particular conserved positions should be targeted for mutation or that alanine substitutions at the specific sites would enhance performance in CRISPR integrated systems.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejections. The Examiner acknowledges the fact that the GenBank entries cited do not teach the variants of SEQ ID NO: 25-26. However, the Examiner disagrees with Applicant’s contention that the variants of SEQ ID NO: 25-26 are not obvious over the prior art.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
With regard to the argument that the teachings of Ma et al. in isolation cannot render obvious the claimed PcrA variants with mutations at different positions from different organisms in a fundamentally different application context, it is noted that the teachings of Ma et al. have not been analyzed in isolation without taking into consideration the subject matter of the claims. The teachings of Mandell and Kong et al. as extensively discussed above render a system for helicase-dependent amplification that comprises two CRISPR/Cas complexes, each binding a different strand of a target nucleic acid, a PcrA helicase, a primer pair and a polymerase obvious. One of skill in the art would reasonably conclude that since Kong et al. teach that a PcrA helicase can be used for amplification, this reference clearly suggests that any PcrA helicase can be used for unwinding double stranded nucleic acids in an amplification method. Ma et al. teach making mutations to a helicase to increase its helicase activity, which is highly desirable in applications that require a helicase.
While it is agreed that Ma et al. did not examine UvrD mutations for integration with CRISPR systems or isothermal amplification applications, the issue in the instant case is whether the proteins of SEQ ID NO: 25-29 would have been obvious PcrA helicases at the time of the invention. Please note that the enzymatic activity of a helicase is the same regardless of its intended use. The proteins of SEQ ID NO: 25-29 are PcrA helicases that are variants of PcrA helicases known in the prior art, namely those disclosed by GenBank accession No. WP_003870487.1, GenBank accession No. WP_049660019.1, GenBank accession No. WP_034654680.1, GenBank accession No. WP_095390358.1 and GenBank accession No. WP_055343022.1. These variants differ from those disclosed in the prior art only by two substitutions at two contiguous positions that substitute a D residue with an alanine. As previously indicated, Ma et al. teach that the E. coli UvrD double mutant D403A/D404A is a protein with hyper-helicase activity. Ma et al. teach that these mutations (UvrD303) favor the closed conformation and will lead to better unwinding activity. Ma et al. teach that UvrD belongs to the SF1 family, such as the PcrA and Rep helicases, and that these helicases have a four-subdomain tertiary arrangement that includes two RecA-like domains (1A/2A) and a flexible domain (2B), believed to have a regulatory role in helicase activity and be a molecular switch. Ma et al. teach that the tilted state and related motions disclosed can possibly help connect structural information with function for other SF1 helicase. Ma et al. teach that PcrA is highly homologous to UvrD and that the low-FRET “open” conformation of PcrA could be similar to the tilted conformation of UvrD. Ma et al. teach that the D403A/D404A substitutions in the 2B domain stabilizes the UvrD closed conformation and that their analysis provides potential target residues to guide future experimental designs to obtain UvrD-like helicases with tunable unwinding activities.
While Applicant states that the rejections assume obviousness without adequate motivation, it is noted that in view of the teachings of Ma et al. and the specific reference to the structural similarity between the E. coli UrvD helicase and PcrA helicases, including certain structural conformations, one of skill in the art would have been highly motivated to make the two substitutions that correspond to the substitutions D403A/D404A in the E. coli UrvD helicase of Ma et al. in the PcrA helicases of GenBank accession No. WP_003870487.1, GenBank accession No. WP_049660019.1, GenBank accession No. WP_034654680.1, GenBank accession No. WP_095390358.1 and GenBank accession No. WP_055343022.1 to obtain variants having hyper-helicase activity to use in any application that uses a helicase, such as an amplification method, which is the subject matter of the claimed invention. As shown in the alignments previously provided, making the two substitutions that correspond to substitutions D403A/D404A in the E. coli UrvD helicase of Ma et al. to the PcrA helicases of GenBank accession No. WP_003870487.1, GenBank accession No. WP_049660019.1, GenBank accession No. WP_034654680.1, GenBank accession No. WP_095390358.1 and GenBank accession No. WP_055343022.1 result in proteins that comprise SEQ ID NO: 25-29, respectively.
While Applicant states that the claimed sequences represent engineered variants with strategic mutations specifically for CRISPR-integrated systems, it is noted that the helicase in the method of Mandell and Kong et al. does not require to interact with the Cas enzyme or the guide RNA of the CRISPR system to unwind DNA, nor does the Cas enzyme or guide RNA require a helicase to form a complex and bind to a target nucleic acid. This is not the case where hyper-helicase activity is required for proper interaction with a Cas enzyme or a guide RNA. Therefore, the motivation to obtain variants with hyper-helicase activity is not limited to their use with a CRISPR system.
With regard to the argument that the prior art does not provide guidance that these particular conserved positions should be targeted for mutation or that alanine substitutions at the specific sites would enhance performance in CRISPR integrated systems, it is noted that the prior art strongly suggests the substitutions of these consecutive D residues with alanine for increasing helicase activity, which is the activity that is required in the method of Mandell and Kong et al. As previously indicated, the helicase in the method of Mandell and Kong et al. is required for unwinding a double stranded nucleic acid and not for the Cas protein to interact with the guide RNA or for binding of the Cas/guide RNA complex to the target nucleic acid. Therefore, contrary to Applicant’s assertions, the PcrA helicases of SEQ ID NO: 25-29 are deemed obvious and functional equivalents of the PcrA helicases of the method of Mandell and Kong et al. As such, the claimed invention is deemed obvious over the cited prior art.
Conclusion
No claim is in condition for allowance.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a). A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to DELIA M RAMIREZ, Ph.D., whose telephone number is (571) 272-0938. The examiner can normally be reached on Monday-Friday from 8:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert B. Mondesi, can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
/DELIA M RAMIREZ/Primary Examiner, Art Unit 1652
DR
February 6, 2026