USE OF ENHANCED POL THETA ACTIVITY FOR EUKARYOTIC GENOME ENGINEERING

DETAILED ACTION 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 . Continued Examination Under 37 CFR 1.114 A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 06/29/2026 has been entered. Restrictions/Elections Applicants’ amendment of claims submitted on 06/29/2026 has been acknowledged. Claims 1, 4-8, 12-15, 18-20 are pending. Claims 4, 5, 7, 12-15, 18-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 07/18/2025. Therefore, claims 1, 6 and 8, along with elected species of SEQ ID NO: 2, the amino acid sequence of Beta vulgaris Pol θ are examined in this office action. Objections/Rejections that are withdrawn 35 U.S.C. 112 rejection has been withdrawn in light of applicant’s amendment of claim 1 to recite the plant is maize plant. 35 U.S.C. 102 rejection has been withdrawn in light of applicant’s cancellation of claim 16. Drawings The drawings are objected to because Figure 7 shows the percentage (%) in parenthesis on the GEMT201, GEMT202 and GEMT248, it is not clear what does the percentage represent?, which has not been described in description of figure of page 9, lines 20-21. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance. Following 35 USC § 112 –Scope of Enablement has been modified in light of applicant’s amendment of claims 1, 6 and 8 to recite the plant is a maize plant and applicant’s cancellation of claim 11. Claim Rejections - 35 USC § 112 –Scope of Enablement Claims 1, 6 and 8 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method comprising providing the at least one plant cell to be modified, upregulating or overexpressing Pol θ as SEQ ID NO:2, introducing at least one genome editing system and a double stranded DNA insert and cultivating the plant cell, does not reasonably provide enablement for the method would increase targeted insertion of any DNA insert in at least one target genomic sequence in any plant cell and the promotion of Pol θ activity increases the DNA insertion efficiency. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims. An “analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.” MPEP 2164.01. “A conclusion of lack of enablement means that. . . the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention [i.e. commensurate scope] without undue experimentation.” In re Wright, 999 F.2d 1557,1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993); MPEP 2164.01. In In re Wands, 858 F.2d 731,8 USPQ2d 1400 (Fed. Cir. 1988), several factors implicated in determination of whether a disclosure satisfies the enablement requirement and whether any necessary experimentation is “undue” are identified. These factors include, but are not limited to: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 858 F.2d 731,737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988). No single factor is independently determinative of enablement; rather “[i]t is improper to conclude that a disclosure is not enabling based on an analysis of only one of the above factors while ignoring one or more of the others.” MPEP 2164.01. Likewise, all factors may not be relevant to the enablement analysis of any individual claim. The Breadth of the Claims and nature of invention: Claim 1 is drawn to a method of increasing targeted insertion in any plant cells by promoting, upregulating or overexpressing Pol θ or a POLQ sequence encoding the same as consisting of SEQ ID NO:2. The state of the prior art: Mara et al. (Published: 2019, Journal: New Phytologist (2019) 222: 1380–1391 doi: 10.1111/nph.15680) teaches POLQ effecting Alt-EJ are involved in the repair of CRISPR-Cas9- induced DSBs in plants (page 1389, right paragraph 2). Mara et al. teaches sequence homology searches of the P. patens genome identified putative homologs for the P. patens POLQ gene different species (Fig. S2) (Phytozome #Pp3c5_12930). Tol et al. (Published: 2022, The Plant Journal 109: 112–125) teaches Pol θ deficiency also prevented ectopic gene targeting (GT) events, suggesting that the sequence of events leading up to this outcome requires TMEJ (page 112, Summary). Sharief et al. teaches there are at least eight human DNA polymerase θ (page 90, Abstract). Jing et al. teaches in the sample of 68 protein sequences of DNA polymerase theta comprising different eukaryotes, three distinct clades of plant, animal and fungal species were found (page 6, Figure 1). Therefore, there is large variation in the structures and function of the Pol θ proteins in different eukaryotic species. Kamoen et al. teaches their finding of the Polymerase theta (Polθ)-mediated end joining (TMEJ) in Arabidopsis showed species-specific functionality of Polθ and point to functional divergence in TMEJ across species (page 1, abstract). Kamoen et al. teaches Polθ-independent MMEJ occur, often requiring longer microhomologies referred to as extended microhomology (eMH) (page 2, left paragraph 3, page 4, left paragraph 5). Kamoen et al. teaches, in plants, the helicase domain can also function independently to facilitate eMMEJ wherein when the flanking sequences contain sufficient homology, Polθ’s polymerase activity is not required when flanking sequences contain sufficient homology (page 5, right paragraph 4, Figure 5). Kamoen et al. teaches Polθ’s polymerase activity becomes obsolete when primer already exist for polymerase delta in TMEJ process (page5, right paragraph 2). Sfeir et al. (Published: 2015, Journal: Trends in Biochemical Sciences, 40 (11): 1-14) (Included in IDS submitted on 01/12/2024) teaches various protein effecting eukaryotic end joining (page 703, see Table 1 below) that are involved in the end joining pathway. This showed various mechanism of targeted insertion. Therefore, the specific effect of the upregulating or overexpression of Pol θ proteins would also depend on other pathways in end joining mechanisms. PNG media_image1.png 911 866 media_image1.png Greyscale Therefore, there is no predictability that the upregulation or overexpression of Pol θ or a POLQ sequence encoding the same as consisting of SEQ ID NO:2 would increase the targeted insertion in any plant cell. Furthermore, Tijsterman et al. (US Patent application Pub. No.: US 2019/0390208 A1, Pub. Date: 12/26/2019) teaches efficient method for gene targeting by reducing (eliminating and avoiding) random integration of transfected DNA wherein the transfected nucleic acid is a nuclease or a component of Crispr/Cas system (page 2, paragraphs 0020-0021). Tijsterman et al. teaches the reduction in POLQ expression and / or activity reduces random integration of transfected nucleic acid molecule in plant cell (claim 1). Tijsterman et al. teaches the reducing expression of POLQ sequence from plants for example sugar beet Beta vulgaris: XP_010667709 which has 100% sequence identity to applicant’s SEQ ID NO: 2 (see enclosed PDF). Therefore there is no evidence that upregulation or overexpression or promotion of DNA Pol θ or a POLQ sequence encoding the same as consisting of SEQ ID NO:2 would increase the targeted insertion in any plant cell. Alignment of applicant’s elected SEQ ID NO:2 to the uniport database showed the sequence has 100% sequence identity to the locus A0A0J8CX06_BETVV as Helicase and polymerase-containing protein TEBICHI disclosed (see alignment below) disclosed by Dohm et al. (see below). Alignment of SEQ ID NO: 2 to uniport database: RESULT 1 A0A0J8CX06_BETVV ID A0A0J8CX06_BETVV Unreviewed; 2133 AA. AC A0A0J8CX06; DT 14-OCT-2015, integrated into UniProtKB/TrEMBL. DT 14-OCT-2015, sequence version 1. DT 02-APR-2025, entry version 48. DE RecName: Full=Helicase and polymerase-containing protein TEBICHI {ECO:0008006|Google:ProtNLM}; GN ORFNames=BVRB_2g031660 {ECO:0000313|EMBL:KMT18137.1}; OS Beta vulgaris subsp. vulgaris (Beet). OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae; OC Caryophyllales; Chenopodiaceae; Betoideae; Beta. OX NCBI_TaxID=3555 {ECO:0000313|EMBL:KMT18137.1, ECO:0000313|Proteomes:UP000035740}; RN [1] {ECO:0000313|EMBL:KMT18137.1, ECO:0000313|Proteomes:UP000035740} RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. RC TISSUE=Taproot {ECO:0000313|EMBL:KMT18137.1}; RX PubMed=24352233; DOI=10.1038/nature12817; RA Dohm J.C., Minoche A.E., Holtgrawe D., Capella-Gutierrez S., Zakrzewski F., RA Tafer H., Rupp O., Sorensen T.R., Stracke R., Reinhardt R., Goesmann A., RA Kraft T., Schulz B., Stadler P.F., Schmidt T., Gabaldon T., Lehrach H., RA Weisshaar B., Himmelbauer H.; RT "The genome of the recently domesticated crop plant sugar beet (Beta RT vulgaris)."; RL Nature 505:546-549(2014). CC --------------------------------------------------------------------------- CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms CC Distributed under the Creative Commons Attribution (CC BY 4.0) License CC --------------------------------------------------------------------------- DR EMBL; KQ090038; KMT18137.1; -; Genomic_DNA. DR EnsemblPlants; KMT18137; KMT18137; BVRB_2g031660. DR Gramene; KMT18137; KMT18137; BVRB_2g031660. DR KEGG; bvg:104884718; -. DR eggNOG; KOG0950; Eukaryota. DR OMA; FHNMCQQ; -. DR OrthoDB; 2320933at2759; -. DR Proteomes; UP000035740; Chromosome 2. DR GO; GO:0005524; F:ATP binding; IEA:UniProtKB-KW. DR GO; GO:0003677; F:DNA binding; IEA:InterPro. DR GO; GO:0003887; F:DNA-directed DNA polymerase activity; IEA:InterPro. DR GO; GO:0051301; P:cell division; IEA:EnsemblPlants. DR GO; GO:0006261; P:DNA-templated DNA replication; IEA:InterPro. DR GO; GO:0006302; P:double-strand break repair; IEA:TreeGrafter. DR GO; GO:1990067; P:intrachromosomal DNA recombination; IEA:EnsemblPlants. DR GO; GO:0009933; P:meristem structural organization; IEA:EnsemblPlants. DR GO; GO:0009640; P:photomorphogenesis; IEA:EnsemblPlants. DR GO; GO:2000011; P:regulation of adaxial/abaxial pattern formation; IEA:EnsemblPlants. DR GO; GO:1902749; P:regulation of cell cycle G2/M phase transition; IEA:EnsemblPlants. DR GO; GO:0010468; P:regulation of gene expression; IEA:EnsemblPlants. DR CDD; cd18026; DEXHc_POLQ-like; 1. DR CDD; cd08638; DNA_pol_A_theta; 1. DR CDD; cd18795; SF2_C_Ski2; 1. DR FunFam; 1.10.150.20:FF:000002; DNA polymerase I; 1. DR FunFam; 1.10.3380.20:FF:000003; Helicase and polymerase-containing protein TEBICHI; 1. DR FunFam; 1.20.1060.10:FF:000003; Helicase and polymerase-containing protein TEBICHI; 1. DR FunFam; 3.40.50.300:FF:000968; Helicase and polymerase-containing protein TEBICHI; 1. DR FunFam; 3.40.50.300:FF:001000; Helicase and polymerase-containing protein TEBICHI; 1. DR Gene3D; 1.10.3380.20; -; 1. DR Gene3D; 3.30.70.370; -; 1. DR Gene3D; 1.10.150.20; 5' to 3' exonuclease, C-terminal subdomain; 1. DR Gene3D; 3.40.50.300; P-loop containing nucleotide triphosphate hydrolases; 2. DR Gene3D; 3.30.420.10; Ribonuclease H-like superfamily/Ribonuclease H; 1. DR Gene3D; 1.20.1060.10; Taq DNA Polymerase, Chain T, domain 4; 1. DR InterPro; IPR011545; DEAD/DEAH_box_helicase_dom. DR InterPro; IPR001098; DNA-dir_DNA_pol_A_palm_dom. DR InterPro; IPR043502; DNA/RNA_pol_sf. DR InterPro; IPR002298; DNA_polymerase_A. DR InterPro; IPR014001; Helicase_ATP-bd. DR InterPro; IPR001650; Helicase_C-like. DR InterPro; IPR046931; HTH_61. DR InterPro; IPR027417; P-loop_NTPase. DR InterPro; IPR048960; POLQ-like_helical. DR InterPro; IPR012337; RNaseH-like_sf. DR InterPro; IPR036397; RNaseH_sf. DR PANTHER; PTHR10133; DNA POLYMERASE I; 1. DR PANTHER; PTHR10133:SF62; DNA POLYMERASE THETA; 1. DR Pfam; PF00270; DEAD; 1. DR Pfam; PF00476; DNA_pol_A; 1. DR Pfam; PF00271; Helicase_C; 1. DR Pfam; PF20470; HTH_61; 1. DR Pfam; PF21099; POLQ_helical; 1. DR PRINTS; PR00868; DNAPOLI. DR SMART; SM00487; DEXDc; 1. DR SMART; SM00490; HELICc; 1. DR SMART; SM00482; POLAc; 1. DR SUPFAM; SSF56672; DNA/RNA polymerases; 1. DR SUPFAM; SSF52540; P-loop containing nucleoside triphosphate hydrolases; 1. DR SUPFAM; SSF53098; Ribonuclease H-like; 1. DR SUPFAM; SSF158702; Sec63 N-terminal domain-like; 1. DR PROSITE; PS51192; HELICASE_ATP_BIND_1; 1. DR PROSITE; PS51194; HELICASE_CTER; 1. PE 4: Predicted; KW ATP-binding {ECO:0000256|ARBA:ARBA00022840}; KW Nucleotide-binding {ECO:0000256|ARBA:ARBA00022741}; KW Reference proteome {ECO:0000313|Proteomes:UP000035740}. FT DOMAIN 501..693 FT /note="Helicase ATP-binding" FT /evidence="ECO:0000259|PROSITE:PS51192" FT DOMAIN 733..925 FT /note="Helicase C-terminal" FT /evidence="ECO:0000259|PROSITE:PS51194" FT REGION 1..42 FT /note="Disordered" FT /evidence="ECO:0000256|SAM:MobiDB-lite" FT REGION 138..187 FT /note="Disordered" FT /evidence="ECO:0000256|SAM:MobiDB-lite" FT REGION 1304..1332 FT /note="Disordered" FT /evidence="ECO:0000256|SAM:MobiDB-lite" FT COMPBIAS 1311..1323 FT /note="Low complexity" FT /evidence="ECO:0000256|SAM:MobiDB-lite" SQ SEQUENCE 2133 AA; 234489 MW; 5260825D4DB7B510 CRC64; Query Match 100.0%; Score 10944; Length 2133; Best Local Similarity 100.0%; Matches 2133; Conservative 0; Mismatches 0; Indels 0; Gaps 0; Qy 1 MDSDEPQTRIDQFFASKKKRALSPKTAKDSSPSAKGSLENYLVTSQNDSVKRSLALEIDL 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1 MDSDEPQTRIDQFFASKKKRALSPKTAKDSSPSAKGSLENYLVTSQNDSVKRSLALEIDL 60 Qy 61 SSKNEKKEIFLPSDGATRGLILPEAIPVDKYGAGNVGGAGNRDNSDQVQEIENVGLKQFA 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 61 SSKNEKKEIFLPSDGATRGLILPEAIPVDKYGAGNVGGAGNRDNSDQVQEIENVGLKQFA 120 Qy 121 LDFLSLYCRRIQTSSLSEHKTHEHKRCGSPSVLAVEGTPSKRRHFVADKENSQPGQETPS 180 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 121 LDFLSLYCRRIQTSSLSEHKTHEHKRCGSPSVLAVEGTPSKRRHFVADKENSQPGQETPS 180 Qy 181 SDKGDSDLVLCSNVTQKVERDVNSALGPQISLRKCSKVTNSTSLVDECSTPAAICKSSSA 240 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 181 SDKGDSDLVLCSNVTQKVERDVNSALGPQISLRKCSKVTNSTSLVDECSTPAAICKSSSA 240 Qy 241 RETPVSTRGSSIFSPGEAFWNEAIQVVDGLFPQTGNNFFQVAEDTGISKTQVVEVFSHCG 300 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 241 RETPVSTRGSSIFSPGEAFWNEAIQVVDGLFPQTGNNFFQVAEDTGISKTQVVEVFSHCG 300 Qy 301 EGSSNNKCDVPVGIYASNTKQAEFTITSPKTHKGILEREASPLPVKHFDFSCDDKNLDES 360 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 301 EGSSNNKCDVPVGIYASNTKQAEFTITSPKTHKGILEREASPLPVKHFDFSCDDKNLDES 360 Qy 361 MPGRVSSGNREDYRDASERAVCNVDHKRPLDDNAVLIGSDVQSHALALKVLDETAPVFGA 420 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 361 MPGRVSSGNREDYRDASERAVCNVDHKRPLDDNAVLIGSDVQSHALALKVLDETAPVFGA 420 Qy 421 EKMRHSVVGPDTDNNISASPNRKNANFVAPGMFDGNSTPSSSMPLEDRLNLSNWLPPAIC 480 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 421 EKMRHSVVGPDTDNNISASPNRKNANFVAPGMFDGNSTPSSSMPLEDRLNLSNWLPPAIC 480 Qy 481 SIYRRKGIAKLYPWQVDCLQMDGVLQKRNLVYCASTSAGKSFVAEILMLRRVIDSGKIAL 540 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 481 SIYRRKGIAKLYPWQVDCLQMDGVLQKRNLVYCASTSAGKSFVAEILMLRRVIDSGKIAL 540 Qy 541 LVLPYVSICVEKAEHLEVLLEPLGKQVRSYYGNQGGGTLPKDTSVAVCTIEKANSLVNRL 600 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 541 LVLPYVSICVEKAEHLEVLLEPLGKQVRSYYGNQGGGTLPKDTSVAVCTIEKANSLVNRL 600 Qy 601 LEEDRLSEVGIIVIDELHMVADPSRGYLLELLLTKLRYGAGEGNGESHSGDSSATSSGKG 660 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 601 LEEDRLSEVGIIVIDELHMVADPSRGYLLELLLTKLRYGAGEGNGESHSGDSSATSSGKG 660 Qy 661 DLTQGLQIVGMSATMPNVAAVADWLHAALYQTEFRPVPLEEFIKVGNNIYDKKMEIVRTV 720 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 661 DLTQGLQIVGMSATMPNVAAVADWLHAALYQTEFRPVPLEEFIKVGNNIYDKKMEIVRTV 720 Qy 721 PKLANLGGKDPDHVVELCNEVVEDGHSVLIFCSSRKGCESTARHISMYLKKFSAKIQEES 780 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 721 PKLANLGGKDPDHVVELCNEVVEDGHSVLIFCSSRKGCESTARHISMYLKKFSAKIQEES 780 Qy 781 SEFADVFSAIDSLRRCPVGLDPVLEETLPSGVAYHHAGLTVEEREIVETCYRKGLVRVLT 840 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 781 SEFADVFSAIDSLRRCPVGLDPVLEETLPSGVAYHHAGLTVEEREIVETCYRKGLVRVLT 840 Qy 841 ATSTLAAGVNLPARRVIFRQPKIGRDFIDGTRYRQMSGRAGRTGIDAKGESVLICRPEEV 900 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 841 ATSTLAAGVNLPARRVIFRQPKIGRDFIDGTRYRQMSGRAGRTGIDAKGESVLICRPEEV 900 Qy 901 KRIKALLNDSCPPLQSCLSEDKNGMTHAILEVVAGGIVQTANDIHRYVRCTLLNSTKPFE 960 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 901 KRIKALLNDSCPPLQSCLSEDKNGMTHAILEVVAGGIVQTANDIHRYVRCTLLNSTKPFE 960 Qy 961 DVVKSAQDSLRWLCHKKFLEWNDDTKLYSTTPLGRAAFGSSLSPEESLIVLDDLSRAREG 1020 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 961 DVVKSAQDSLRWLCHKKFLEWNDDTKLYSTTPLGRAAFGSSLSPEESLIVLDDLSRAREG 1020 Qy 1021 FVLASDLHLVYLATPINVEVEPDWELFYERFMELSSLDQSVGNRVGVIEPFLMRMAHGAP 1080 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1021 FVLASDLHLVYLATPINVEVEPDWELFYERFMELSSLDQSVGNRVGVIEPFLMRMAHGAP 1080 Qy 1081 VRTASRSRNAIKGLHSRLDFQLGVSKSNTLSDEQSLRVCRRFYVALILSRLVQELPVGEV 1140 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1081 VRTASRSRNAIKGLHSRLDFQLGVSKSNTLSDEQSLRVCRRFYVALILSRLVQELPVGEV 1140 Qy 1141 CAAFKVARGMVQSLQENAGRFASMVSLFCERLGWFDLEGLVAKFQNRVSFGVRAEIVELT 1200 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1141 CAAFKVARGMVQSLQENAGRFASMVSLFCERLGWFDLEGLVAKFQNRVSFGVRAEIVELT 1200 Qy 1201 TIPYVKGSRARALFKAGLRTPLAIA EASVTEIAKALFESLSWAQEDLAQRRVQLGIAKKI 1260 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1201 TIPYVKGSRARALFKAGLRTPLAIA EASVTEIAKALFESLSWAQEDLAQRRVQLGIAKKI 1260 Qy 1261 KNAARKIVLDKAEEARVAAFSAFQSLGLNVPQLSRPVLLPASGIPIDNDESTPSSGEDST 1320 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1261 KNAARKIVLDKAEEARVAAFSAFQSLGLNVPQLSRPVLLPASGIPIDNDESTPSSGEDST 1320 Qy 1321 SSSFHQKRKHMSVELVERQGNEGDPGTDAGRPAKKLVTSLEISTERKAVNTLPSGVGVIR 1380 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1321 SSSFHQKRKHMSVELVERQGNEGDPGTDAGRPAKKLVTSLEISTERKAVNTLPSGVGVIR 1380 Qy 1381 DSSTPIQDSVPVGVMTLSPCLGQSASVASVGIVNKEDQRWETNNAGSDGNKKLSNDKGPI 1440 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1381 DSSTPIQDSVPVGVMTLSPCLGQSASVASVGIVNKEDQRWETNNAGSDGNKKLSNDKGPI 1440 Qy 1441 NIASTSGLESLLDLWESVGEFYFDIHFNKKSEANSLLAYEVHGLAICWEDSRVYYVNLAK 1500 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1441 NIASTSGLESLLDLWESVGEFYFDIHFNKKSEANSLLAYEVHGLAICWEDSRVYYVNLAK 1500 Qy 1501 ILQNSNFGSCNIECSGHVNPIDANNLLFTENLLWNRISRLLGRVGVRKFTWNLKLQVKVL 1560 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1501 ILQNSNFGSCNIECSGHVNPIDANNLLFTENLLWNRISRLLGRVGVRKFTWNLKLQVKVL 1560 Qy 1561 KNAVLSLNKNNLSQTLKSTTIEVADGSFCKLPQVILQDGIDLSIVAWILWPDEERSSNPS 1620 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1561 KNAVLSLNKNNLSQTLKSTTIEVADGSFCKLPQVILQDGIDLSIVAWILWPDEERSSNPS 1620 Qy 1621 LEKEVKRRLSSEAAGAANQSGRWKNQMRRAAHNGCCRRAAQTRALHSVLWKLLVSEELVE 1680 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1621 LEKEVKRRLSSEAAGAANQSGRWKNQMRRAAHNGCCRRAAQTRALHSVLWKLLVSEELVE 1680 Qy 1681 ALLGVELPLVHVLADMEDQGIGVDMEGCLKARHVLSKKLKILEKEAYKLAGMMFSLYMPA 1740 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1681 ALLGVELPLVHVLADMEDQGIGVDMEGCLKARHVLSKKLKILEKEAYKLAGMMFSLYMPA 1740 Qy 1741 DIANVLYGHLKLPIPEGHNKGKQHASTDKHCLDLLRLEHPIVPVIKEHRTLAKLLNCTLG 1800 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1741 DIANVLYGHLKLPIPEGHNKGKQHASTDKHCLDLLRLEHPIVPVIKEHRTLAKLLNCTLG 1800 Qy 1801 SICSLAKLSMRTQRYTVHGRWLQTSTATGRLSMEEPNLQCVEHMVEFRMHQNGDAQCDSD 1860 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1801 SICSLAKLSMRTQRYTVHGRWLQTSTATGRLSMEEPNLQCVEHMVEFRMHQNGDAQCDSD 1860 Qy 1861 GTDYKINARDFFIPTEENWLLVTADYSQIELRLMAHFSKDPSLIELLTDPHGDAFTMIAS 1920 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1861 GTDYKINARDFFIPTEENWLLVTADYSQIELRLMAHFSKDPSLIELLTDPHGDAFTMIAS 1920 Qy 1921 RWTGKLESVVSSQERDQTKRLVYGILYGMGANTLAEQLNCSADEAKAKIQSFKNSFPGVA 1980 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1921 RWTGKLESVVSSQERDQTKRLVYGILYGMGANTLAEQLNCSADEAKAKIQSFKNSFPGVA 1980 Qy 1981 SWLHEAVAFCRQHGYVQTLKGRKRFLSKIKIGDSKEKAKAQRQAVNSICQGSAADIIKIA 2040 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 1981 SWLHEAVAFCRQHGYVQTLKGRKRFLSKIKIGDSKEKAKAQRQAVNSICQGSAADIIKIA 2040 Qy 2041 MINIYSVIAGCETRSSNDALVAKFSTLKGHCRILLQVHDELVMEVDPSVLGEAGLLLKTC 2100 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Db 2041 MINIYSVIAGCETRSSNDALVAKFSTLKGHCRILLQVHDELVMEVDPSVLGEAGLLLKTC 2100 Qy 2101 MESAASLSVPLQVKVKFGRTWGSLEPFNVNDLR 2133 ||||||||||||||||||||||||||||||||| Db 2101 MESAASLSVPLQVKVKFGRTWGSLEPFNVNDLR 2133 The amount of direction provided by the inventor: Applicant describes SEQ ID NO: 2 is a Beta vulgaris amino acid sequence (see sequence listing). The existence of working examples: The Specification or state of the art does not teach a person with skill in the art how to make and/or use the subject matter within the full scope of these claims because: Applicant fails to teach promoting or upregulating or overexpression of Pol θ as SEQ ID NO: 2 as a Beta vulgaris amino acid sequence would increase targeted insertion in maize plant cells. Applicant has zero example of upregulating or overexpression of Pol θ as SEQ ID NO: 2 as a Beta vulgaris amino acid sequence or any other Pol θ would increase targeted insertion in maize cells. Lack of a working example is a critical factor to be considered, especially in a case involving an unpredictable and undeveloped art.  See MPEP § 2164. Genetech, 108 F.3d at 1366, states that “a patent is not a hunting license.  It is not a reward for search, but compensation for its successful conclusion” and “[p]atent protection is granted in return for an enabling disclosure of an invention, not for vague intimations of general ideas that may or may not be workable”. In the absence of guidance from either the instant disclosure or the art, it would require undue trial and error experimentation for a skilled artisan to make and use the Pol θ as SEQ ID NO: 2 to know whether it will increase targeted insertion of a DNA insert, with no reasonable expectation of success in arriving at the invention of the method leading to the increased targeted insertion in the recited maize plant cells. Given the large variations of pathways leading to targeted insertion of a DNA insert in a plant and various effects of specific Pol θ genes and their effects in a maize plants for increasing targeted insertion of a DNA insert and specificity of the Pol θ genes, specifically, does not provide predictability in increasing targeted insertion in maize plant cells by a method of promoting or upregulating or overexpressing Pol θ as SEQ ID NO: 2 as a Beta vulgaris amino acid sequence. Instead, applicant has zero example of such increased targeted insertion by promoting or upregulating or overexpressing Pol θ as SEQ ID NO: 2 as a Beta vulgaris amino acid sequence or by any other Pol θ in any plant. Thus, in view of the unpredictability associated with the effect of the Pol θ as SEQ ID NO: 2 as a Beta vulgaris amino acid sequence for increasing targeted insertion in maize plants and also in sugar beet (Beta vulgaris) itself, the lack of enabling guidance from either the instant disclosure or the art, and breath and diversity of the embodiments encompassed by the claimed genus, the lack of sufficient working examples, and the level of the art at the time of the invention, one of ordinary skill in the art must rely on undue trial and error experimentation to make and test the numerous polypeptides encompassed by the broad genera, in order to make and/or use the invention within the full scope of these claims. For at least this reason, the Specification does not teach a person with skill in the art how to make and/or use the subject matter within the full scope of these claims. Response to Argument Applicants’ arguments filed 06/29/2026 have been fully considered but they are not persuasive. Applicant argues claim 1 is amended to recite that the plant is a maize plant. Applicant submits that the Examples discuss that overexpressing Pol 8 in maize increases targeted insertion. See Example 4 and Figs. 6-7 (Response to Rejection, page 8, paragraph 5). Applicant's arguments have been fully considered but they are not persuasive since Example 4 teaches the construct overexpressing ZmPolQ (SEQ ID NOs: 180 (plasmid)) which would be from maize and it does not show the evidence that there was increase in targeted insertion of a DNA insert. Figures 6 and 7 only shows the targets sites in the sequence and a cartoon of the target sites. Since applicant recites SEQ ID NO: 2 which is from Beta vulgaris amino acid sequence, there is unpredictability associated with the method of introducing such amino acid and upregulating its expression would increase targeted insertion of a DNA insert in the maize plant including maize. Furthermore, in paragraph [0018]-[0020] of the Application as published only states the finding of Pol θ activation was surprising without any evidence the activation of SEQ ID NO:2 would have any surprising results. Therefore, the scope of enablement rejection has been maintained. Response to Argument Applicants’ arguments filed 12/29/2025 have been fully considered but they are not persuasive. Applicant argues Applicant herein amends claim 1 to incorporate the subject matter of claim 3, which is not rejected over either Gomez or Mara for this reason Gomez or Mara fails to anticipate the claim. Applicant's arguments have been fully considered but they are not persuasive since the amendment of claim 1 does not change the rejection on claim 16 which is a product of a plant cell that would not require the method steps since the claim has been analyzed as product by process, see above. Summary No claim is allowed. Claims 1, 6 and 8 are free of a prior art. The closest prior art is Mara et al. that discloses a method of producing P. patens plants produced by the method for increasing targeted insertion of a DNA insert by promoting DNA polymerase theta (Pol θ) activity in the plant cell and by introducing gene editing system CRISPR/Cas9 in the plant cell and regenerating the plant cell. Mara et al. does not specifically teach increasing targeted insertion by promoting protein of amino acid sequence of SEQ ID NO:2, as a protein sequence from Beta vulgaris subsp. vulgaris (see alignment above). Since there are many polymerase theta sequences and for example Kamoen et al. teaches their finding of the Polymerase theta (Polθ)-mediated end joining (TMEJ) in Arabidopsis showed species-specific functionality of the Polθ and Kamoen et al. point to functional divergence in TMEJ across species (page 1, abstract), there is no predictability that the promotion of Polθ as SEQ ID NO:2 would increase targeted insertion in any plant cells (see Scope of enablement rejection above). Examiner’s Contact Information Any inquiry concerning this communication or earlier communications from the examiner should be directed to SANTOSH SHARMA whose telephone number is (571)272-8440. The examiner can normally be reached Mon-Fri 8:00 AM - 5:00 PM. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, AMJAD A. ABRAHAM can be reached at (571)270-7058. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /SANTOSH SHARMA/Examiner, Art Unit 1663 /DAVID H KRUSE/Primary Examiner, Art Unit 1663