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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 02/27/2026 has been entered.
Status of the Applications, Amendments and/or Claims
This action is written in response to applicant's correspondence(s) submitted on 02/04/2026. In the paper of 02/04/2026, Applicant amended claims 1, 4 and 11 and canceled claim 6. Accordingly, claims 1, 3-5 and 7-20 are pending. Claims 3, 7-8 and 12-20 are still withdrawn, non-elected with traverse. Claims 1, 4-5 and 9-11 remain under review.
Concerning withdrawn claim 7, Applicant is put on notice that the Office does not permit a claim to be dependent from a canceled claim i.e. claim 7 depends from canceled claim 6.
Response to Arguments
Moot and/or Withdrawn Rejection(s)
The objection to the specification for failing to provide proper antecedent basis for the subject matter of claim 1 because the specification does not disclose alteration of a target site polynucleotide within planta is withdrawn based on amendments that are newly made to the preamble of claim 1.
The rejections of claims 1, 4-5 and 9-11 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite as stated in paragraphs 9-10 of pages 3-4 of the Final rejection of 11/14/2025, because the preamble of claim 1 recites the limitation “alteration of target site polynucleotide within planta” and no definition provided in the specification for what is intended by “target site polynucleotide” are withdrawn based on amendments that are newly made to the preamble of claim 1.
The rejections of claim 6 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite as stated in paragraphs 9-10 of pages 3-4 of the Final rejection of 11/14/2025, because the preamble of claim 1 recites the limitation “alteration of target site polynucleotide within planta” and no definition provided in the specification for what is intended by “target site polynucleotide” are moot based on the cancellation of claim 6.
The rejection of claim 6 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite because claim 1, step (d) recites a step of identifying at least one nucleotide insertion, deletion, substitution, or modification of the sequence of the target site polynucleotide after the insertion event of step (c), yet, claim 1 omits an essential element i.e. the rationale for why alternative modifications that are beyond an insertion event, such as deletion or substitution, are being recited in claim 1, step (d) is moot based on the cancellation of claim 6.
The rejection of claim 6 under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends is now moot based on the cancellation of claim 6.
Maintained Rejection(s)
The rejection of claims 1, 4-5 and 9-11 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite because claim 1, step (d) recites a step of identifying at least one nucleotide insertion, deletion, substitution, or modification of the sequence of the target site polynucleotide after the insertion event of step (c), yet, claim 1 omits an essential element i.e. the rationale for why modifications that are beyond an insertion event, such as deletion or substitution, are being recited in claim 1, step (d) is maintained.
The examiner believes claim 1, step (d) does not sufficiently indicate the origin for the at least one nucleotide insertion, deletion, substitution, or mutation at the target site of the polynucleotide results from the insertion event of claim 1, step (c) i.e. at present, claim, step (c) does not qualify that the identifying performed in step (d) is not directed to sequencing the insertion site of the target polynucleotide for the presence of the newly inserted heterologous donor polynucleotide comprising of at least one nucleotide insertion, deletion, substitution, or mutation;
but rather that the identifying step (d), is directed to identifying nucleotide insertion(s), deletion(s), substitution(s), or mutation(s) resulting from inserting heterologous donor polynucleotide via a double strand break repair at the double strand break site of the target polynucleotide, wherein the double strand break repair is selected from homologous recombination repair, or non-homologous end joining repair.
Claim Objections
Claim 1 is objected to because of the following informalities: Claim 1, step (c) recites the limitation “within the double-strand break of the target site of the polynucleotide: and,”. The colon within the phrase “the polynucleotide:” within this specific limitation, should be replaced with a semicolon and the comma at the end of the phrase “and,” of the limitation should be deleted.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 4-5 and 9-11 are 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The preamble of claim 1 recites the limitation “a method of alteration of a target site of a polynucleotide within a plant cell”. This phrase “within a plant cell” within the preamble limitation is found to lack clarity, thus indefinite as it is unclear if “within a plant cell” involves an in-vitro editing process for a template DNA of a plant cell; or intends an in-vivo editing of plant cell DNA, particularly since it is unclear to the Office how the step of identifying at least one nucleotide insertion, deletion, substitution or modification as claimed in claim 1, step(d) would be performed in-vivo.
None of the processes recited by claim 1(a)(i)-1(a)(iii) through claim 1(c) and recited by claim 9 indicate where the polynucleotide editing of claims 1 and 9 occurs (in-vitro and/or in-vivo of the plant cell).
Furthermore, it is unclear how to achieve the limitation of claim 1, step (d) as an in-vivo step and/or how the limitation of claim 1, step (d) accomplishes the preamble as recited by claim 1.
Claims 4-5 and 9-11 are further rejected as they depend from claim 1.
The rejection of claims 1, 4-5 and 9-11 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite because claim 1(c) and the subsequent step (d) are found to omit an essential element.
Claim 1, step (c) recites a step of inserting a cleaved heterologous donor polynucleotide product of a tandemly concatenated repeat unit within a double strand break generated within a target polynucleotide by contact of a Cas-gRNA complex and the target polynucleotide.
Claim 1, step (c) and claim 1, step (d) each appear to omit an essential element.
Concerning claim 1, step (c), step (c) does not specify that inserting of the heterologous donor polynucleotide within the double strand break of the target site of the polynucleotide involves a double strand break repair, and the double strand break repair occurs via homologous recombination repair or non-homologous end joining repair double-strand break site.
Concerning claim 1, step (d), step (d) does not specify that identifying the alteration of the target polynucleotide involves identifying one or more nucleotide insertion(s), deletion(s), substitution(s) derived from the homologous recombination repair, or non-homologous end joining repair i.e. claim 1, step (d) is not directed to identifying nucleotide insertion(s), deletion(s), substitution(s) within the cleaved heterologous donor polynucleotide product that is to be introduced at the target double strand break site.
Claims 4-5 and 9-11 are further rejected as they depend from claim 1.
Claim 9 recites the limitations “wherein the frequency of homologous repair at the double strand break site” and “the rate of non-homologous end joining repair”. Claim 9 depends from claim 1 and claim 1 fails to require any type of double strand break site repair. Accordingly, there is insufficient antecedent basis for the limitations “the frequency of homologous repair” and for “the rate of non-homologous end joining repair” in claim 9.
Closest prior art
The closest prior art references are:
Nelson et al. (1) (US Patent No. 11,834,670, filed April 19, 2017);
Nelson et al. (2) (US2018/0305718A1, pub. April 19, 2017, filed April 19, 2017);
Bak et al. (WO2018/195555A1, pub. Oct 25, 2018, filed April 23, 2018);
Li et al. (pub. Sept 12, 2016, Nat Plants. 2016; 16139, pp. 1-6);
Dahan-Meir et al. (April 18, 2018, The Plant Journal, 95(1), pp.5-16);
Cignan et al. (WO2015/026883A1, pub. Feb 26, 2015);
Anand et al. (CA3097209A1, pub. Nov 14, 2019).
Nelson et al. (1) and (2) both teach site-specific modification of an endogenous target DNA of a eukaryotic cell is provided. The method comprises contacting the endogenous target DNA having an intended modification site with (i) a gene editing system configured to introduce a double strand break in the endogenous target DNA at or near the intended modification site, and (ii) a donor DNA repair template comprising a plurality of tandem repeat sequences.
In the method of Nelson et al., each of the plurality of tandem repeat sequences comprises an exogenous donor DNA sequence flanked by a donor 5′ flanking sequence and a donor 3′ flanking sequence. The donor 5′ flanking sequence and the donor 3′ flanking sequence are homologous to a continuous DNA sequence on either side of the intended modification site in the endogenous target DNA.
The donor DNA repair template is a concatemeric DNA that includes a plurality of tandem repeat sequences, which can repair the endogenous target DNA sequence by inserting the donor DNA repair template at or near the intended modification site. The donor DNA repair template comprises two homology arms, and an exogenous donor DNA sequence. The homology arms of the donor DNA repair template are constructed on either side of the exogenous donor DNA sequence. The homology arms of the donor DNA repair template are referred to herein as donor 5′-flanking sequence and donor 3′-flanking sequence. The 5′ flanking sequence is a left homology arm and the 3′ flanking sequence is a right homology arm. Each of the plurality of tandem repeat sequences in the donor DNA repair template comprises an exogenous donor DNA sequence flanked by a donor 5′ flanking sequence and a donor 3′ flanking sequence.
Bak et al. teach a method for efficiently modifying the genome of human cells via sequential homologous recombination using CRISPR/Cas-mediated genome editing with donor DNA delivered by two or more adeno-associated virus (AAV) vectors. The method of Bak et al. provides a CRISPR/Cas9- based method to enable site-specific integration of large polynucleotide comprising generating a DNA double-stranded break (DSB) at a specific site in the genetic locus and repairs the DSB via sequential homologous recombination (HR) (two-step HR) by using two recombinant adeno- associated viruses (AAVs), each containing a portion of the HR donor template. Sequential or iterative homologous recombination in cells is effective even if the size of the target polynucleotide exceeds the packaging capacity of AAV, which is about 4.5 kb from AAV Inverted Terminal Repeat (ITR) to ITR.
The genomic editing system and method of Bak et al. can seamlessly fuse two portions of a large target polynucleotide together via consecutive homologous recombination events using two different AAV donor vectors containing different donor templates and the CRISPR/Cas9 genomic editing system. In some embodiments, the first donor template contains a first portion of the target polynucleotide and the same sgRNA target site that mediates its integration into the target genetic locus. This sgRNA site is reconstituted in the genome after integration. In the second homologous recombination event, the second donor template fuses a second portion of the target polynucleotide to the first portion using the introduced sgRNA target site.
Li et al. teach an intron-mediated site-specific gene replacement and insertion method that generate mutations using the nonhomologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%.
Li et al. teach targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant.
Dahan Meir et al. teach a method for targeted mutagenesis and gene replacement in tomato using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.
Omitted from Nelson et al., (1) and (2), Bak et al., Li et al. and Dahan-Meir et al.
Nelson et al. and Bak et al. do not teach eukaryotic cell whose genome is altered/ or whose gene is edited is a plant cell; and do not teach “alteration of a target site polynucleotide within a plant cell”; or “cleaving the plurality of sequence units of (a)(iii) with the complex of (a)(ii), releasing the heterologous donor polynucleotides” or “inserting the heterologous donor polynucleotide within the double-strand break of the target site polynucleotide”.
Bak et al. and Li et al. do not teach providing to a target site polynucleotide, a tandemly concatenated template composed of a plurality of sequence units, each sequence unit comprising a heterologous donor polynucleotide, wherein each sequence unit is flanked by a set of first flanking sequences, each of which is capable of hybridization with the guide RNA of (a)(ii).
Dahan-Meir et al. do not teach “cleaving the plurality of sequence units of (a)(iii) with the complex of (a)(ii), releasing the heterologous donor polynucleotides” or “inserting the heterologous donor polynucleotide within the double-strand break of the target site polynucleotide”.
Cignan et al. (WO2015/026883A1)
Cignan et al. is directed to a genome modification of a target sequence in the genome of a plant or plant cell. The method of Cignan et al. introduces a guide RNA/Cas endonuclease system into said plant cell to alter target sites within the genome of a plant, plant cell or seed. The guide RNA and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site.
Cignan et al. also teach method comprising introducing a guide RNA and a donor DNA into a plant cell having a Cas endonuclease, wherein said guide RNA and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site, wherein said donor DNA comprises a polynucleotide of interest. The method comprises identifying at least one plant cell that has a modification at said target, wherein the modification includes at least one deletion or substitution of one or more nucleotides in said target site.
Cignan et al. teach introducing the guide RNA or donor DNA directly by particle bombardment or via Agrobacterium transformation of a recombinant DNA construct. Cignanet al. teach recombinant DNA construct comprising the corresponding guide DNA operably linked to a plant U6 polymerase III promoter.
Cignan et al. teach in Figure 20, a schematic of a donor DNA (also referred to as HR repair DNA) comprising a transgene cassette with a selectable marker (phosphomannose isomerase, depicted in grey), flanked by homologous recombination sequences (HR1 and HR2) of about 0.5 to 1 kb in length, used to introduce the transgene cassette into a genomic target site for the guide RNA Cas endonuclease system.
Cignan et al. teach a DD20CR1 guide RNA Cas9 complex transcribed from the linked guide RNA/Cas9 DNA cassettes will cleave specifically the DD20CR1 target site on chromosome 04 to make DNA double strand breaks. The breaks can be repaired spontaneously as NHEJs or repaired as a HR event by the donor DNA facilitated by the flanking homologous regions DD20-HR1 and DD20HR2.
Anand et al. (CA3097209A1)
Anand et al. teach a method comprising improving homology-directed repair of a double strand break in a plant cell, via the use of a polynucleotide comprising sequences homologous to the target site. Anand et al. teach the double strand break is created by an RNA-guided Cas endonuclease. The homology-directed repair of the double-strand break may include incorporation of a heterologous polynucleotide, for example a gene encoding a trait of agronomic importance. The homology-directed repair of the double-strand break may occur as a result of template-directed repair using a polynucleotide repair template.
Omitted from Cignan et al. (WO2015/026883A1) and Anand et al. (CA3097209A1)
Cignan et al. and Anand et al. do not teach a donor DNA that is composed of a plurality of sequence units provided as tandemly concatenated repeats, wherein each sequence unit comprises a heterologous donor polynucleotide and wherein each sequence unit is flanked by a set of first flanking sequences, each of which is capable of hybridization with the guide RNA of (a)(ii).
Any comments considered necessary by applicant must be submitted no later than the payment of the issue fee and, to avoid processing delays, should preferably accompany the issue fee. Such submissions should be clearly labeled “Comments on Statement of Reasons for Allowance.”
Conclusion
No claims are currently allowed.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to OLAYINKA A OYEYEMI whose telephone number is (571)270-5956. The examiner can normally be reached Monday -Thursday: 9:00 am - 5:00 pm, EST.
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OLAYINKA A. OYEYEMI
Examiner
Art Unit 1681
/OLAYINKA A OYEYEMI/Examiner, Art Unit 1681
/GARY BENZION/Supervisory Patent Examiner, Art Unit 1681
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