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 .
This action is in response to the amendment filed 10/28/2025, in which claims 1, 6, 9, 10, 11 and 13 were amended and claims 2-5, 7, 8 and 12 were canceled. Claims 1, 6, 9-11 and 13 are currently pending.
Applicant’s arguments have been thoroughly reviewed, but are not persuasive for the reasons that follow. Any rejection and objections not reiterated in this action have been withdrawn. This action is FINAL.
Priority
Acknowledgment is made of applicant's claim for foreign priority under 35 U.S.C. 119(a)-(d). Receipt of the certified copy of the foreign priority document, CN2019/107731937, is acknowledged.
Acknowledgment is made of applicant's claim to foreign priority document CN2019/107731937 filed on 08/21/2019.
All claims are given the priority date of 08/21/2019.
Claim Objections
The previous claim objections of claims 1 and 2 are withdrawn in view of Applicant’s amendments to the claims filed on 10/28/2025.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
This is a previous rejection rewritten necessitated by the amendment to the claims filed on 10/28/2025.
Claim 13 is rejected 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. Claim 13 require editing of a single gene, specifically the claim recites “when a single gene is edited”. However, claim 13 depends from claim 10 which requires editing of at least two genes. Specifically claim 10 recites “comprising target sequences of genes affecting a spore color change, and target sequences of genes not affecting phenotypes of Aspergillus”. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Response to Amendments - Claim Rejections - 35 USC § 112
The previous rejection of claims 12 and 13 under 35 USC 112(b) has been withdrawn in view of Applicant’s amendments filed on 10/28/2025.
The previous rejection of claim 5 under 35 USC 112(d) is moot in view of Applicant’s amendments filed on 10/28/2025 in which claim 5 was canceled.
The previous rejection of claim 13 under 35 USC 112(d) has been maintained and rewritten in view of Applicant’s amendments filed on 10/28/2025.
Applicants’ arguments have been considered but are not found persuasive. Applicant argues that the office states that claim 13 requires the editing of a single gene and that reconsideration and withdrawal of the rejection is requested.
Applicant’s arguments are not persuasive because, as stated above, claim 13 depends from claim 10 wherein claim 10 requires the editing of two genes such as a gene effecting spore color change as well as a gene not effecting a phenotype. However, claim 13 recites “when a single gene is edited”. Therefore, claim 13 does not further limit and/or require all the limitation from the claim upon which it depends.
Response to Amendments - Claim Rejections - 35 USC § 102
The previous rejection of claims 1 and 10 under 35 USC 102(a)(1)/(a)(2) as being anticipated by Meijrink et al (WO 2016/110453 A1) has been withdrawn in view of Applicant’s amendments to the claims as filed on 10/28/2025.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 10 and 11 are rejected under 35 U.S.C. 103 as being unpatentable by Meijrink et al (WO 2016/110453 A1; cited in previous office action) in view of Song et al (PLoS ONE, 13(8), e0202868, Pgs. 1-17 and Supplemental information’ August 24, 2018; cited in previous office action). This is a NEW rejection necessitated to address the amendment to the claims in the reply filed 10/28/2025.
Regarding claim 10, the independent claim 10 is interpreted as a system with an intended use of gene knockout.
Meijrink teaches a method for visualized screening of an Aspergillus strain with gene editing, comprising a plasmid using CRISPR-Cas9 gene editing technology to simultaneously knock out genes (a) and (b) in the Aspergillus; wherein (a) is a gene affecting a spore color change, such as fwnA and (b) is a second gene, such as nicB (Page 13, lines 14 bridging Page 14, line 3; and Page 105, Example 20). Meijrink teaches the plasmid comprising a Cas9 gene encoding the protein, the gRSR cassette comprising the fwnA gene and nicB gene and dsRED which is a detectable fluorescent signal (Page 32, Lines 17-20 and Page 185, Figure 25). Meijrink teaches the use of a gRSR cassette (a single guide RNA cassette with flanking ribozymes (Page 3, Lines 1-5 and Page 161, Fig. 2) comprising the fwnA gene and nicB gene sequences (Page 107, Lines 1-19 and Page 185, Fig. 25). The knockout of nicB results in no change in the phenotype when the Aspergillus is cultured in the presence of nicotinamide (Page 105, Example 20).
Meijrink does not teach that the sgRNA expression cassette encodes an Aspergillus endogenous tRNA, such as tRNAAla, tRNAArg, or tRNAPhe.
Song teaches CRISPR/Cas9 systems applied in filamentous fungi to improve the efficiency of genome alteration (Page 1, Abstract). Song teaches a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence (Page 1, Abstract). Song teaches co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger, therefore the results demonstrate that tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger (Page 1, Abstract). Song teaches the tRNAs are listed in supplemental table 1 including tRNAA1a, tRNAArg, tRNAPhe and tRNAIle (Supplemental Table 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Meijrink to include the aminoacyl tRNAs as taught by Song because Meijrink teaches it is within the ordinary skill in the art to use a method for visualized screening of an Aspergillus strain with gene editing, wherein using CRISPR-Cas9 gene editing technology to simultaneously knock out genes (a) and (b) in the Aspergillus; wherein (a) is a gene affecting a spore color change, such as fwnA and (b) is a second gene, such as nicB, wherein the knockout of nicB results in no change in the phenotype when the Aspergillus is cultured in the presence of nicotinamide and Song teaches cotransformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger.
One would have been motivated to make such a modification in order to receive the expected benefit of tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger as taught by Song.
Regarding claim 11, Meijrink teaches a method where the CRISPR-Cas systems according to the invention can be used to target multiple target sites, where two or more guide polynucleotides are provided together with the CRISPR-Cas system to allow the formation of two or more CRISPR-Cas complexes for multiplex editing (Page 38, lines 11-25; Page 91, lines 27-29). Meijrink exemplifies a method of multiplex editing of two target sites in a single step in an Aspergillus strain, comprising using CRISPR-Cas9 gene editing technology to simultaneously knock out genes (a) and (b) in the Aspergillus; wherein (a) is a gene affecting a spore color change, such as fwnA and (b) is a second gene, such as nicB (Page 13, lines 14 bridging Page 14, line 3; and Page 105, Example 20). The knockout of nicB results in no change in the phenotype when the Aspergillus is cultured in the presence of nicotinamide (Page 105, Example 20). Meijrink teaches that other genes may be targeted, preferably amy A, preferably of Aspergillus niger (Page 57, lines 21-27). Meijrink teaches the amy A has a nucleotide sequence as set forth in instant SEQ ID NO:52 showing 100% identity to the A. niger CBD513.88 sequence of SEQ ID NO: 1 (Page 57, Lines 21-29; See Appendix I, mailed 7/29/2025).
Meijrink does not teach amyA as the second gene that is knocked out.
It would have been obvious to one of ordinary skill in the art before the effective filing
date of the claimed invention to modify the method of multiplex editing of two genes in
Aspergillus of Mejirink to substitute editing of nicB as the second gene with editing of amyA as
the second gene, because Meijrink teaches it is within the ordinary skill in the art to use the
CRISPR-Cas system with two or more guide polynucleotides to simultaneously target two genes,
including fwnA, and teach that it is preferable to knock out amyA. The substitution of one gene
for another would lead to the predictable knockout of known genes taught by Mejirink.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable by Meijrink et al (WO 2016/110453 A1; cited in previous office action) in view of Song et al (PLoS ONE, 13(8), e0202868, Pgs. 1-17 and Supplemental information’ August 24, 2018; cited in previous office action), as applied to claims 10 and 11 above, and further in view of Maiyuran et al (WO 2019/046703 A1; cited in previous office action). This is a NEW rejection necessitated to address the amendment to the claims in the reply filed 10/28/2025.
The teachings of Meijrink and Song are applied and discussed above.
Regarding claim 13, Meijrink teaches the use of a U6 RNA polymerase III promoter (Page 35, lines 5-8 and Page 11, Line 26). Meijrink teaches the use of a U6 terminator (Page 11, Line 27).
Meijrink does not teach the use of the specific aminoacyl-tRNAs required by the instant claims.
Song teaches CRISPR/Cas9 systems applied in filamentous fungi to improve the efficiency of genome alteration (Page 1, Abstract). Song teaches a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence (Page 1, Abstract). Song teaches co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger, therefore the results demonstrate that tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger (Page 1, Abstract). Song teaches the tRNAs are listed in supplemental table 1 including tRNAA1a, tRNAArg, tRNAPhe and tRNAIle (Supplemental Table 1). Song teaches the tRNAA1a1 sequence which is 100% identical to instant SEQ ID NO: 10 (Appendix III, mailed 7/29/2025). Song teaches the tRNAArg2 sequence which is 100% identical to instant SEQ ID NO: 20 (Appendix IV, mailed 7/29/2025). Song teaches the use of a U6 promoter is most widely used (Page 9, Paragraph 1). Song teaches the sgRNA structure showing the RNA promoter followed by the (generic) tRNA wherein the tRNA could be any of the tRNAs cited (Page 5, Figure 1B).
Meijrink and Song do not teach specifically a first tRNA after a promoter is tRNAAla, and a second tRNA is tRNAArg as well as Pu6 promoter has the nucleotide sequence as set forth in SEQ ID NO:3 and the Tu6 terminator has the nucleotide sequence as set forth in SEQ ID NO:5.
Maiyuran teaches a Pu6 promoter and a Tu6 terminator are used to separately promote
and terminate an expression of sgRNA (Page 2, Lines 23-30). Maiyuran teaches the Pu6
promoter that has a nucleotide sequence as set forth in instant SEQ ID NO: 3 is 100% identical to
SEQ ID NO: 33 (Page 21, Lines 17-18; See Appendix VII, mailed 7/29/2025). Maiyuran teaches the Tu6 terminator that has a nucleotide sequence as set forth in instant SEQ ID NO:5 is 100% identical to SEQ ID NO: 34 (Page 23, Lines 6-7; See Appendix VIII, mailed 7/29/2025). Maiyuran teaches the M. oryzae U6-2 promoter, shown as SEQ ID NO: 33, increased the transformation efficiency dramatically (Page 55, Lines 28-30 bridging Page 56, Lines 1-5). Maiyuran teaches the addition of the M. oryzae U6-2 terminator, shown as SEQ ID NO: 34, improved the function of the CRISPR/Cas9 system dramatically, improving the transformation efficiency approximately 6-fold (Page 56, Lines 22- 25). Maiyuran teaches the structure of the sgRNA cassette where the tRNAgly is after the U6-2 promoter (Page 110, Fig. 13).
It would have been obvious to one of ordinary skill in the art before the effective filing
date of the claimed invention to modify the teachings of Meijrink and Song to include a Pu6
promoter and a Tu6 terminator as taught by Maiyuran as well as to substitute the tRNAgly of Maiyuran for the tRNAAla first after the promoter and a second tRNA is tRNAArg because Meijrink teaches it is within the ordinary skill in the art to use a method for visualized screening of an Aspergillus strain with gene editing, wherein using a plasmid comprising a Cas9 gene encoding the protein, the gRSR cassette comprising the fwnA gene and nicB gene and dsRED which is a detectable fluorescent signal to simultaneously knock out a gene affecting a spore color change, such as fwnA and a second gene, such as nicB, wherein the knockout of nicB results in no change in the phenotype when the Aspergillus is cultured in the presence of nicotinamide wherein two or more guide polynucleotides are provided together with the CRISPR-Cas system to allow the formation of two or more CRISPR-Cas complexes for multiplex editing, Song teaches the sgRNA structure showing the RNA promoter followed by the (generic) tRNA wherein the tRNA could be any of the tRNAs such as the sequences for tRNAAla1 and tRNAArg2 to generate the intended mutation in A. niger and Maiyuran teaches the M. oryzae U6-2 promoter and terminator improved function of the CRISPR/Cas9 system dramatically as well as the structure of the sgRNA cassette where the tRNAgly is after the U6-2 promoter.
One would have been motivated to make such a modification in order to receive the
expected benefit of the two guide polynucleotides with one comprising the structure of the U6-2 promoter followed by the tRNAAla1 and the second comprising the structure of the U6-2 promoter followed by the tRNAArg2 for the multiplex editing of the Aspergillus as taught by Meijrink, Song and Maiyuran.
Response to Arguments - Claim Rejections - 35 USC § 103
The previous rejection claims 2, 3, 9 and 11 under 35 USC 103 as being obvious by Meijrink et al (WO 2016/110453 A1) has been withdrawn in view of Applicant’s cancelation and amendments of the claims as filed on 10/28/2025.
The previous rejection claims 4-7 under 35 USC 103 as being obvious by Meijrink et al (WO 2016/110453 A1) in view of Song et al (PLoS ONE, 13(8), e0202868, pgs. 1-17 and Supplemental information; August 24, 2018) has been withdrawn in view of Applicant’s cancelation and amendments of the claims as filed on 10/28/2025.
The previous rejection claims 8 and 12 under 35 USC 103 as being obvious by Meijrink et al (WO 2016/110453 A1) in view of Song et al (PLoS ONE, 13(8), e0202868, pgs. 1-17 and Supplemental information; August 24, 2018) as applied to claims 1-7, 9 and 11 above, and further in view of Maiyuran et al (WO 2019/046703 A1) is moot in view of Applicant’s cancellation of the claims on 10/28/2025.
The previous rejection of claim 13 under 35 USC 103 as being obvious by Meijrink et al (WO 2016/110453 A1) in view of Song et al (PLoS ONE, 13(8), e0202868, pgs. 1-17 and Supplemental information; August 24, 2018) as applied to claims 1-7, 9 and 11 above, and further in view of Maiyuran et al (WO 2019/046703 A1) has been withdrawn in view of Applicant’s amendments of the claims as filed on 10/28/2025.
Applicant’s amendments to the claims have overcome the previous rejections of record and thus a new rejection have been made above as necessitated by amendments to the claims filed on 10/28/2025.
Conclusion
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.
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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637