Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1, 3, 5-12, 14-16 and 18-21 are pending and being examined.
All previous objections and rejections not set forth below have been withdrawn in view of applicant’s amendments to the claims. However, the claim amendments by the Applicant necessitated new prior art references and new grounds of rejections, as discussed below.
Claim Rejections - 35 USC § 103
Claims 1, 3, 5-12, 14-16 and 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over Begemann et al. (US 9896696 B2, published on 02/20/2018) in view of GenBank accession No. RHA18620.1 (published on 6 October 2018) and in evidence of Zhang et al. (Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems, 2021, Nat. Commun., 12:1944; published on 29 March 2021).
Begemann et al. describes a method of modifying a nucleotide sequence at a target site in a genome (abstract) by using a Cpf1 (also known as Cas12a1) polypeptide (abstract). Begemann et al. also describes a DNA-targeting RNA (gRNA) (column 38, line 31-32, claim 8) which interacts with the Cas12a polypeptide and forming a complex (as recited in claim 1) to direct the Cas12a/Cpf1 endonuclease to a specific target site (column 3, last 2 lines; and column 4, first line), as recited in claim 11. It describes the DNA-targeting RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in the target DNA, and (b) a second segment that interacts with a Cpfl polypeptide (column 37, last 2 lines; column 38 first 5 lines), as recited in claim 11. The method taught by Begemann et al. is applicable to plant cells and plant embryos (column 4, line 22-25).
Begemann et al. describes that the method produces a staggered double stranded breaks (DSBs) (column 4, line 16-17) (as recited in claim 6) at pre-determined target sites resulting in modified nucleotide sequence (as recited in claim 5) in form of mutation, insertion, and/or deletion of DNA sequences (as recited in claim 7) at the target site(s) in a genome (abstract) including the genome of a plant cell (column 4, line 22-25), as recited in claim 7, including in a monocotyledonous plant rice (Oryza sativa) (as recited in claims 8-9) (column 2, line 29-31; Fig. 1-3; column 15, line 33; column 15, line 36; column 28, line 27; column 43, line 64). It is known in the art that the modification of the target sequence especially insertion(s) is/are provided by the heterologous (gRNA) sequence, as recited in claim 7. Begemann et al. also explicitly describes insertion of heterologous genes (read on to DNA) (as recited in claim 7) and modification of native plant gene expression to achieve desirable plant traits (column 21, line 30-32).
Begemann et al. teaches many promoters including regulated, growth stage-specific, or tissue-specific promoters (column 14, line 1-2); a constitutive promoter like maize ubiquitin promoter (column 2, line 38-39); and a promoter operable in cells of interest (abstract, line 8-9), which reads on to “a cell type-specific promoter”, as recited in claim 10. Begemann et al. teaches target sites located immediately 3' of a PAM site in the genome of the plant cell, and wherein the PAM site comprises TTTV (where V represents adenine (A), cytosine (C), or guanine (G); as commonly acknowledged in the art) (Fig. 2A-B; Fig. 3A-B; Fig. 4B-D; Fig. 5A; column 24, line 20; column 58, line 6), as recited in claim 12.
Begemann et al. describes a polynucleotide sequence encoding codon optimized Cas12a (or Cpf1) polypeptide for expression in a plant cell (column 9, line 14-17), as recited in claim 15. It is known in the art that Cas12a polypeptides remain active, albeit of varying degree, in the temperature range from 22°C to 37°C (Zhang et al., page 3, left column, para 3, line 14-18), as recited in claim 14.
Regarding claim 16, Begemann et al. describes that the nuclease domain of Cas12a/Cpf1 comprise a RuvC-like domain (column 5, line 20-21) which can be inactivated (column 4, line65-67). Specific mutation including deletion of/in the RuvC domain or specific substitution(s) in the Cas12a polypeptide (column 5, line 32-34) can make nuclease deactivated Cas12a protein (dCas12a).
Begemann et al. describes the polynucleotide encoding the Cas12a polypeptide present in a vector (column 13, line 44-45), as recited in claim 20, and regenerating a plant from the (genome edited or transformed) plant cell (column 29, line 23-25), as recited in claim 21.
However, Begemann et al. does not describe a Cas12a polypeptide having at least 95% sequence identity to instant SEQ ID NO: 23.
GenBank accession No. RHA18620.1 teaches a type V CRISPR-associated protein Cas12a/Cpf1 from Eubacterium ventriosum comprising at least 95% (98%) sequence identity to instant SEQ ID NO: 23, as shown below.
Title: US-18-558-934-23
Perfect score: 6435
Sequence: 1 MESNNKIFTETIGTSSIAKT..........KELKIRHQDWFDFIQNKRYL 1234
Database : AASEQ2_04072026_151950.fasta:*
RESULT 1
AASEQ2_04072026_151950
Best Local Similarity 98.0%; Query Match 99.7%; Score 6412.5; DB 1; Length 1259;
Matches 1234; Conservative 0; Mismatches 0; Indels 25; Gaps 1;
Qy 1 MESNNKIFTETIGTSSIAKTMRNSLVPTESTKRNIEKNGIIIDDQLRAEKRQQLKEIMDE 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MESNNKIFTETIGTSSIAKTMRNSLVPTESTKRNIEKNGIIIDDQLRAEKRQQLKEIMDE 60
Qy 61 YYRTYIDNKLSNVALTRTIDWKELFQAIEDNYKQNTTKTKNELEKKQKEKRTEIYKILSD 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 YYRTYIDNKLSNVALTRTIDWKELFQAIEDNYKQNTTKTKNELEKKQKEKRTEIYKILSD 120
Qy 121 DEKFKQLFNAKLLTNVLPEFIKNQNIDNEEKQEKISTVELFQRFTSSFTDFFKNRKNVFS 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 DEKFKQLFNAKLLTNVLPEFIKNQNIDNEEKQEKISTVELFQRFTSSFTDFFKNRKNVFS 180
Qy 181 KDEISTSICYRVVQENAWIFYQNLLAFEEI-------------------------EIFDF 215
|||||||||||||||||||||||||||||| |||||
Db 181 KDEISTSICYRVVQENAWIFYQNLLAFEEIKKTAEQEIEKIEAENRDSISDYSLKEIFDF 240
Qy 216 DFYGLLLNQGGIRFYNDVCGKINYHMNLYGQKHNIKSNKFKMKRMHKQILSIDESTFEVP 275
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 DFYGLLLNQGGIRFYNDVCGKINYHMNLYGQKHNIKSNKFKMKRMHKQILSIDESTFEVP 300
Qy 276 TMFENDKEVYQVLNEFLSDLASKKILERVEKIGENVSEYEINKIYIQSKNFEKFSSFMCG 335
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 TMFENDKEVYQVLNEFLSDLASKKILERVEKIGENVSEYEINKIYIQSKNFEKFSSFMCG 360
Qy 336 NWQIINDSLKTYYNEKIKSKGKAKEEKVKKAIKAIEYKSLADINQLVERYNNDELNRKAE 395
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 NWQIINDSLKTYYNEKIKSKGKAKEEKVKKAIKAIEYKSLADINQLVERYNNDELNRKAE 420
Qy 396 EYISAINEKIKDLDVNEIEYDEKINLIENETKSEEIKSKLDSIMEIMHWTKMFIIEEEIE 455
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 EYISAINEKIKDLDVNEIEYDEKINLIENETKSEEIKSKLDSIMEIMHWTKMFIIEEEIE 480
Qy 456 KDVNFYNEIEEIYDELQPLVTIYNRIRNYVTQKPYSEEKIKLNFGIPTLANGWSKTKEYD 515
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 KDVNFYNEIEEIYDELQPLVTIYNRIRNYVTQKPYSEEKIKLNFGIPTLANGWSKTKEYD 540
Qy 516 NNAIIMIRDGKYYLGIFNAKNKPDKKIMEGHQSEENGDYKKMIYRLLPGPNKMLPKVFMS 575
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 NNAIIMIRDGKYYLGIFNAKNKPDKKIMEGHQSEENGDYKKMIYRLLPGPNKMLPKVFMS 600
Qy 576 KTGIAEYKPSQYILECYEQNKHIKSDKNFDIKFCRDLIDFFKTSINRHPEWSKFNFKFSE 635
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 KTGIAEYKPSQYILECYEQNKHIKSDKNFDIKFCRDLIDFFKTSINRHPEWSKFNFKFSE 660
Qy 636 TSEYEDISTFYREVEKQGYKIEWTYISEKEIKELDENGQLYLFQIYNKDFSEKSKGKENL 695
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 TSEYEDISTFYREVEKQGYKIEWTYISEKEIKELDENGQLYLFQIYNKDFSEKSKGKENL 720
Qy 696 HTMYLKNLFSEENLKNIVLKLNGEAEVFFRKSSIKKPIIHKKGSVLVNKTYNENGERKSI 755
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 HTMYLKNLFSEENLKNIVLKLNGEAEVFFRKSSIKKPIIHKKGSVLVNKTYNENGERKSI 780
Qy 756 PEEQYTEIYKYLNSIGTNELSEKSKKLMEEGKVEYYKANYDIVKDYRYSVDKFFIHLPMT 815
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 PEEQYTEIYKYLNSIGTNELSEKSKKLMEEGKVEYYKANYDIVKDYRYSVDKFFIHLPMT 840
Qy 816 INFKAAGFSPINNIALKNIALKDDMHIIGIDRGERNLIYVSVIDTKGNIVEQRNFNIVNG 875
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 INFKAAGFSPINNIALKNIALKDDMHIIGIDRGERNLIYVSVIDTKGNIVEQRNFNIVNG 900
Qy 876 IDYKEKLKQKELDRDNARKNWKEIGKIKDLKEGYLSLVVHEIAKLVVKYNAIITMEDLNQ 935
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 IDYKEKLKQKELDRDNARKNWKEIGKIKDLKEGYLSLVVHEIAKLVVKYNAIITMEDLNQ 960
Qy 936 GFKRGRFKVERQVYQKFETMLINKLNYLVDKDLAVDQEGGLLRGYQLTYIPESLKVLGRQ 995
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 GFKRGRFKVERQVYQKFETMLINKLNYLVDKDLAVDQEGGLLRGYQLTYIPESLKVLGRQ 1020
Qy 996 CGYIFYVPAAYTSKIDPTTGFVAIFNYKGMTDKDFVTSFDSIKYDDERGLFAFEFDYENF 1055
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 CGYIFYVPAAYTSKIDPTTGFVAIFNYKGMTDKDFVTSFDSIKYDDERGLFAFEFDYENF 1080
Qy 1056 VTHKVEMARNKWTVYTYGERIKRKFKNGSWDTAEKVDLTYQMRSILEKYEIEYNKGQDIL 1115
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 VTHKVEMARNKWTVYTYGERIKRKFKNGSWDTAEKVDLTYQMRSILEKYEIEYNKGQDIL 1140
Qy 1116 EQIEELDEKAQNGICKEIKYLVKDIVQMRNSLPDNAAEDYDAIISPVINNNGEFFDSTRG 1175
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 EQIEELDEKAQNGICKEIKYLVKDIVQMRNSLPDNAAEDYDAIISPVINNNGEFFDSTRG 1200
Qy 1176 DEDKPLDADANGAYCIALKGLYEVMQIKKNWNEETEFPRKELKIRHQDWFDFIQNKRYL 1234
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 DEDKPLDADANGAYCIALKGLYEVMQIKKNWNEETEFPRKELKIRHQDWFDFIQNKRYL 1259
It would have been obvious to one with ordinary skill in the art to use a functional equivalent of the Cas12a endonuclease described by Begemann et al. There are several Cas12a proteins available and known in the art. Using any functional equivalent of the Cas12a, as described by Begemann et al., including the Cas12a from Eubacterium ventriosum (EvCas12a), as described in GenBank accession No. RHA18620.1, would have been an experimental design choice of the artisan.
The Artisan would have been motivated to use to Ev1Cas12a to modify a target site in a plant genome.
Claims 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Begemann et al. (US 9896696 B2) in view of GenBank accession No. RHA18620.1 as applied to claims 1, 3, 5-12, 14-16 and 20-21 above, and further in view of Swartjes et al. (Editor’s cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a, 2019, Biochemical Society Transactions, 48:207–219).
Claim 18 depends from claim 1 and is drawn to a Cas12a polypeptide fused to a deaminase domain modifying or editing a base at the target nucleotide sequence. Claim 19 also depends from claim 1 and is drawn to a Cas12a polypeptide fused to a reverse transcriptase domain, and wherein the DNA-targeting RNA is a prime editing guide RNA (pegRNA).
Begemann et al. in view of GenBank accession No. RHA18620.1 describe a method of modifying a nucleotide sequence at a target site in the genome of a plant cell by introducing into the plant cell- (i) a Casl2a polypeptide, having at least 95% (98%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and (ii) a DNA-targeting RNA capable of forming a complex with the Casl2a polypeptide and directing the complex to the target site, as discussed above. Begemann et al. describe that the nuclease activity of a Cas12a protein can be converted into nickases or be rendered catalytically inactive by conserved residues in the catalytic sites in the RuvC domain of the Cas12a protein, as discussed above, or by deleting the RuvC domain altogether.
However, Begemann et al. do not explicitly describe a Cas12a polypeptide fused to a deaminase domain and/or a reverse transcriptase domain.
Swartjes et al. describes that active nucleases like Cas9 and Cas12a can be converted into nickases or be rendered catalytically inactive by mutating conserved residues in the catalytic sites (page 213, para 7, line 1-2). Swartjes et al. teaches programmable DNA editing for different applications comprising base editing and prime editing (abstract). It describes that Cas nucleases can direct fused cytidine or adenine deaminase enzymes to specific target DNA sequences (page 213, para 8, line 2-3). Catalytically inactive Cas nucleases fused to cytidine or adenine deaminase enzymes performs conversions of C*G pairs to T*A (using cytidine deaminase), or A*T to G*C (using adenine deaminase) (page 213, para 8, line 4-5). Editing a single base offers several advantages including de-novo site-directed mutagenesis and it overcomes the limitation of PAM site availability for any specific gene by creating new or novel PAM site(s) that a specific Cas endonuclease can recognize (page 214, para 1, line 2-3).
Swartjes et al. also teaches prime editing technique that offers much more versatile method for DSB-independent genome editing (page 214, para 2, line 1-2). The technique uses a nickase or nuclease-inactivated Cas endonuclease fused to a reverse transcriptase and a guide RNA that act as a primer (which reads on to “prime editing guide RNA” or “pegRNA”, as recited in claim 19) for the reverse transcriptase enabling introducing small insertions, deletions, and substitutions to be made without non-homologous end-joining (NHEJ) or homologous recombination (HR) (page 214, para 2, line 3-6). Prime editing offers a more control over exact edit (page 214, Table 1).
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to fuse a deaminase domain (of a cytidine or adenine deaminase enzyme), as described by Swartjes et al., with a nuclease inactivated Cas12a protein, as described by Begemann et al. and Swartjes et al., (as recited in claim 18) with a goal to edit any target genomic sequence in a genome without introducing any foreign genetic material. Creating new PAM site(s) at a target gene also expands the possibility of gene editing using a specific Cas endonuclease including Cas12a.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to fuse a deaminase domain with a nuclease inactivated Cas12a protein with a realistic goal to edit any target genomic sequence in a genome without introducing any foreign genetic material. Creating new PAM site(s) at a target gene also expand the possibility of gene editing using a specific Cas endonuclease including Cas12a.
Regarding claim 19, before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan, and the artisan would have been motivated, to fuse a reverse transcriptase or a reverse transcriptase domain (from a reverse transcriptase enzyme) (as described by Swartjes et al.) with a nuclease-inactivated Cas endonuclease (as described by Begemann et al. and Swartjes et al.) and using a pegRNA (as described by Swartjes et al.) with a realistic goal to make the method of Cas based gene editing more versatile and having more control over prime editing to enable small insertions, deletions, and replacements to be made independent from any DNA repair mechanism like NHEJ or HR, as described by Swartjes et al. (page 214, para 2).
Conclusion
No claim is allowed.
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 nonprovisional extension fee (37 CFR 1.17(a)) 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 mailing date of this final action.
Communication
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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1Cas12a is also called Cpf1, as evidenced by Jeon et al. (Direct observation of DNA target searching and cleavage by CRISPR-Cas12a, 2018, Nat Commun., 9, 2777; page 1, abstract).
Prosecution Insights