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 .
Claim Status
Claims 1, 3-28 and 37-41 are pending.
Claims 2 and 29-36 are cancelled by the Applicant.
Claims 37-41 are newly added.
The newly added claims 37-41 would have been included in Group I and, thus, is examined.
Claim 28 is withdrawn from examination as being part of the non-elected invention.
Claims 1, 3-27 and the newly added claims 37-41 are being examined.
All previous objections and rejections not set forth below are withdrawn in view of Applicant’s amendments or arguments.
Examiner’s Note
It is noted by the Examiner that many parts of the response (e.g., bridging paragraph between p.10-11) submitted by the Applicant on 6/17/2026 are not clear to convey the meaning as it is not written in black font but in red, purple, or blue font. The Examiner sees only black fonts and not any color ones. The Applicant is requested to follow the patent filing rule that states to file using "black font" instead filing using colored font in any future submission, as deemed necessary. The Applicant is requested to submit the documents including any response and arguments using black font only with shades of grey highlight, if needed, as per the patent filing rule.
However, as a courtesy to the Applicant and in an effort to provide better customer service, the Examiner continued examining the application.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.821 - 1.825. This application contains a “Sequence Listing” as a PDF file (37 CFR 1.821(c)(2)) or as physical sheets of paper (37 CFR 1.821(c)(3)). A copy of the "Sequence Listing" in computer readable form (CRF) has been submitted; however, the content of the CRF does not comply with one or more of the requirements of 37 CFR 1.822 through 1.824, as indicated in the "Error Report" that indicates the "Sequence Listing" could not be accepted. Refer to attachment or document "Computer Readable Form (CRF) for Sequence Listing – Defective" (CFRD) dated 06/17/2026.
Required response – Applicant must provide:
A replacement "Sequence Listing" part of the disclosure, as described above in item 1); together with
An amendment specifically directing its entry into the application in accordance with 37 CFR 1.825(b)(2);
A statement that the "Sequence Listing" includes no new matter as required by 37 CFR 1.825(b)(5); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(b)(4).
If the replacement "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter and
An amendment to the specification to remove the “Sequence Listing previously submitted as a PDF file (37 CFR 1.821(c)(2)) or as physical sheets of paper (37 CFR 1.821(c)(3))
If the replacement "Sequence Listing" part of the disclosure is submitted according to item 1) c) or d) above, Applicant must also provide:
A CRF in accordance with 1.821(e)(1) or 1.821(e)(2) as required by 37 CFR 1.825(b)(6)(ii); and
Statement according to item 2) a) or b) above.
Claim Objections
Claim 1 is objected to because of the following informalities:
Claim 1 recites “Case9 nickase” in line 3-4. It appears that the Applicant implies “Cas9 nickase”. Appropriate correction is required.
However, “Case9” in the claim is interpreted as “Cas9” for further examination.
Specification
The amendment in the claim listing filed on 6/17/2026 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:
The Applicant has submitted a new sequence listing on 6/17/2026, comprising 70 sequences. Out of the 70 sequences, 22 are new sequences which were not present in previous sequence listing submitted on 5/19/2023 and have no mention or discussion, whatsoever, in the description of the invention initially submitted. This subject matter has not been explicitly, implicitly or inherently described in the originally filed application.
Applicant’s explanation for support for the new sequences (response, p.16-17) citing Fig.16-17 in the drawing submitted on 9/21/2023 could not ascertained by the Examiner. Instant Fig-16-17 does not specify SEQ ID NOs to a sequence mentioned therein and also does not specify or describe each of the 22 new sequences submitted. The application as originally filed fails to support, explicitly, implicitly or inherently, the new sequence listing comprising new sequences.
Applicant is required to cancel the new matter in the reply to this Office Action.
Claim Rejections - 35 USC § 112(a)
Written Description
Claims 7-9 and 12-15 remain 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 claims contain 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Applicant describes, “studies have demonstrated that scRNA formed by adding two MS2 hairpins to the 3′ end of (e)sgRNA can efficiently mediate CRISPRa in human cells, wherein MS2 is a commonly used RNA aptamer. Therefore, the scRNA vector pOsU3-esgRNA-2×MS2 driven by the OsU3 promoter was first constructed” (page 19, line 25-28). Such “scaffold system” of gene editing using scRNA is known in the art (Shakirova et al., Cell Reprogramming With CRISPR/Cas9 Based Transcriptional Regulation Systems, 2020, Front. Bioeng. Biotechnol., 8:882; page 4, left column, para 6, line 1-8). The sequences of 2xMS2 aptamer and the (MS2) aptamer-specific binding protein MCP are standard and long well known in the art ((Zalatan et al. (US 2017/0233762 A1) describes 2xMS2 aptamer sequence (SEQ ID NO: 9) and MCP protein sequence (SEQ ID NO: 2)).
Regarding claims 7-9, the Applicant describes that SEQ ID NO:13 contains a transcription terminator sequence (TTTTTTGTTTTTTATGTCT) (sup4 transcription terminator; response, p.10, para 6; p.11, para 1). This sequence was not described in the originally filed application as comprising a transcription terminator sequence, and the originally filed specification does not mention sup4. Further, transcription terminator sequences would not be in a transcribed RNA and cannot be part of an scRNA. Applicants also state that it is not present at the end of the final transcribed gRNA (response, paragraph bridging p. 10-11). Yet, claim 7 continues to reference a sequence identifier for scRNA (a transcribed RNA) that includes a transcription terminator. Claim 7 depends from claim 1, which does indicate that the claimed genome editing system can comprise an expression construct containing a nucleotide sequence encoding the first scRNA. But claim 1 also indicates the claimed system can comprise a first scRNA itself, and claim 7 refers to the scRNA, not the expression construct encoding it.
Regarding claims 12-13, the Applicant describes that SEQ ID NO:22 contains esgRNA (SEQ ID NO: 4) sequence fused to 2boxB sequence (response, p.11, para 5) and the transcription terminator sequence (TTTTTTGTTTTTTATGTCT) at the 3’ end from position 147 to 165 of SEQ ID NO: 22 (data not shown). The transcription terminator sequence (TTTTTTGTTTTTTATGTCT) cannot be in a transcribed sequence and, thus, cannot be part of the scRNA set forth by SEQ ID NO: 22.
Moreover, the newly submitted sequence of 2boxB (SEQ ID NO: 68) (response, p.17) has 100% sequence identity to esgRNA sequence set forth by SEQ ID NO: 4, as shown below.
Title: US-17-909-309A-4
Perfect score: 106
Sequence: 1 gtttaagagctatgctggaa..........tttttttgttttttatgtct 106
Searched: 1 seqs, 167 residues
Database : NASEQ2_07202026_155401.fasta:*
RESULT 1
NASEQ2_07202026_155401
Best Local Similarity 100.0%;Query Match 81.1%; Score 86; DB 1; Length 167;
Matches 86; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC 80
Qy 61 TTGAAAAAGTGGCACCGAGTCGGTGC 86
||||||||||||||||||||||||||
Db 81 TTGAAAAAGTGGCACCGAGTCGGTGC 106
The boxB sequence and resulting 2boxB sequence as described by the Applicant is also very different (alignment data now shown) than the boxB sequence (GCCCUGAAAAAGGGC) recited in prior art (Said et al., In vivo expression and purification of aptamer-tagged small RNA regulators, 2009, Nucleic Acids Research, 37:e133; Fig. 1B; Supplementary Fig. S1B). The Applicant does not describe how the instant boxB sequence (or 2boxB sequence) is derived.
Considering the breadth of the claims and lack of structure function relationship based on the instant description, the Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Claim Rejections - 35 USC § 102(a)(1)
Claims 1, 4-6, 14-15 and 20 remain rejected under 35 U.S.C. 102(a)(1) as being anticipated by Brezgin et al. (Dead Cas Systems: Types, Principles, and Applications, 2019, Int. J. Mol. Sci., 20, 6041).
Claim 1 is drawn to a multiplex genome editing system in a plant comprising a Cas9 nickase along with one or more scRNAs wherein each scRNA contains an aptamer, a fusion protein that comprises an aptamer-specific binding protein, and a nucleotide base (adenine or cytosine) deamination domain.
Brezgin et al. describes a multiplex genome editing system in various organisms including plants (page 1, para 1, line 2-3) using nickase Cas9 (nCas9) which increases efficiency of base editing (page 12, para 6, line 2-4). Brezgin et al. also describes the “scaffold technique” (page 7, Fig. 2) where modified gRNAs comprising scaffold RNA (scRNA) containing two MS2 aptamers (as recited in claims 4 and 6) (page 3, para 9, line 5-6) and an aptamer-specific binding protein (MCP) (as recited in claim 5) fused to effector molecule(s) resulting in fusion proteins (of aptamer-specific binding proteins and effector proteins). Effectors proteins can be a deaminase, either for adenine or cytosine.
It is well known in the art that aptamers are part of scRNAs (i.e., the scaffold sequence in a gRNA). Brezgin et al. describes simultaneously editing or modulating many genes to control complex biological processes with unprecedented accuracy. This multiplexing is done by a Cas endonuclease that cuts a single RNA transcript into many gRNAs (i.e., paired gRNAs) which would read on to targeting individual targets (page 15, para 4), which can be more than three, and would read on to “first”, “second”, and “third” scRNA and corresponding “first”, “second”, and “third” target regions, as recited, as optional being as part of a Markus group among (ii-1) to (ii-3), in claim 1. Brezgin et al. describes various effector proteins comprising cytidine/cytosine deaminases like APOBEC1 (page 13, para 2, line 1) (as recited in claims 14-15); and adenine deaminases including TadA adenosine deaminase (page 12, para 5, last 2 lines) and ADAR2 adenosine deaminase (page 13, para 4), which are known to comprise adenine deamination domain. TadA is a DNA dependent adenine deaminase (page 12, para 5, line 9-10), as recited in claim 20, that specifically targets DNA (page 15, table 4).
Response to Applicant’s Arguments: The Applicant argues that “… Brezgin only mentions in the base-editing section that nCas9 can be fused with deaminase and UGI to improve editing efficiency (see paragraph 6 on page 12 of Brezgin) but does not teach using nCas9 as the core backbone for multi-editing” (response, p.13, para 6, line 5-7). The Applicant continue to argue, “Brezgin never discloses or teaches "a fusion protein comprising an RNA aptamer-specific binding protein (e.g., MCP) and a cytosine deaminase domain (or adenine deaminase domain)" as instantly claimed” (p.14, para 1, line 1-5). The Applicant contends that “multi-editing as described by Brezgin “does not involve simultaneous different types of editing (base substitution + DSB deletion) in one transformation as instantly claimed (p.14, para 2, last 2 lines). Lastly, the Applicant argues that “the multiple gRNAs in the Cas12a-based gene editing system disclosed in Brezgin target different targets. In contrast, the paired gRNAs employed in the presently claimed subject matter target different strands of DNA in the same third target region (p.14, para 3, line 2-4).
The Examiner disagrees. Brezgin et al. describes several fusion proteins comprising nCas9, which have a single mutated nucleolytic domain and one domain with preserved (DNA) cleavage activity, linked to base-editing factors and co-expressed with UGI (p.12, para 6, line 2-4). Brezgin et al. also describes gRNAs with two MS2-hairpins (aptamers) (p.3, last para, line 6-7) while the two molecules of MCP interact with 1 MS2 hairpin (p.3, last para, line 10). Brezgin et al. teaches that CRISPRa systems can be coupled with affinity-binding technology that enables simultaneous recruitment of multiple domains to the target site (p.6, last para, line 1-2) while modified gRNAs carrying aptamer sequences (MS2, PP7, or com) and attract aptamer-specific proteins (MCP, PCP, Com) fused to transcriptional effector proteins (p.6, last para, line 7-9). Targeting different sites in a genome using specific gRNAs containing the aptamer sequences like MS2 is a well known and routine method in the art (Brezgin et al., p. 11, para 9, line 1-3; Fig. 2).
Moreover, claim 1 recites, “… and ii) one or more or all items selected from the group consisting of: ii-1)….” (line 5). Thus, satisfying at least one condition, i.e., targeting one or the “first target region” would be sufficient to satisfy the claim limitation.
Claim Rejections - 35 USC § 103
Claims 3, 21-26 and 38 are rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above (see rejections under 35 USC §102) further in view of Gao et al. (WO2019120283A1), and in evidence of Delannoy et al. (Arabidopsis tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of Chloroplast tRNAArg(ACG) and Is Essential for Efficient Chloroplast Translation, 2009, Plant Cell, 21:2058–2071) and Chen et al. (Fusion Protein Linkers: Property, Design and Functionality, 2013, Adv Drug Deliv Rev. 2013 October 15; 65:1357–1369).
Claim 38 is drawn to a Cas9 nickase comprising the amino acid sequence of SEQ ID NO: 25, while claim 3 is drawn to a gRNA sequence having 100% sequence identity to instant SEQ ID NO: 3.
Brezgin et al. describes a multiplex genome editing system comprising a Cas9 nickase (nCas9) fused to a (cytosine or adenine) nucleotide deaminase, along with one or more scRNA(s) wherein each scRNA contains an aptamer, a fusion protein that comprises an aptamer-specific binding protein, and a nucleotide base (adenine or cytosine) deamination domain, as described above. Brezgin et al. also describes use of peptide linkers (as recited in claims 23 and 25) to further enhance efficacy of the fusion proteins (page 3, para 6, line 3-5; page 4, para 4, line 3-5).
However, Brezgin et al. does not describe a Cas9 nickase comprising the amino acid sequence of SEQ ID NO: 25 or a polynucleotide sequence set forth in SEQ ID NO: 4.
Gao et al. describes a method for performing efficient base editing to a target sequence in a plant genome by a Cas9-cytidine deaminase fusion protein. It describes simultaneous editing three homoalleles in hexaploid bread wheat conferring heritable resistance to powdery mildew (page 23, para 2, line 2-4). Gao et al. teaches a nuclease-inactivated Cas9 nickase (SEQ ID NO: 3) (page 5, para 0060) comprising 100% identity to instant SEQ ID NO: 25 (as recited in claim 38), as shown below.
RESULT 1
BGM50953
ID BGM50953 standard; protein; 1367 AA.
AC BGM50953;
DT 22-AUG-2019 (first entry)
DE Streptococcus pyogenes Cas9 protein, SEQ ID 3.
KW CRISPR associated 9; CRISPR-Cas9 system; Cas9 protein; crop improvement;
KW genome editing; herbicide resistance; plant breeding; transgenic plant.
OS Streptococcus pyogenes.
CC PN WO2019120283-A1.
CC PD 27-JUN-2019.
CC PF 21-DEC-2018; 2018WO-CN122640.
PR 21-DEC-2017; 2017CN-11393160.
PR 28-APR-2018; 2018CN-10402244.
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
CC PI Gao C, Li C, Zong Y, Wang Y;
DR WPI; 2019-53605Q/53.
CC PT System useful for base editing target sequence in plant genome, comprises base-editing fusion protein, and guide RNA, and expression construct comprising nucleotide sequence encoding base-editing fusion protein, and guide RNA.
CC PS Claim 4; SEQ ID NO 3; 60pp; English.
CC PT System useful for base editing target sequence in plant genome, comprises
base-editing fusion protein, and guide RNA, and expression construct comprising nucleotide sequence encoding base-editing fusion protein, and guide RNA.
CC PS Example 5; Fig 7; 60pp; English.
Query Match 100.0%; Score 6998; Length 1384; Best Local Similarity 100.0%;
Matches 1367; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 2 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 61
Qy 61 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 62 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN 121
Qy 121 IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 122 IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV 181
Qy 181 DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 182 DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL 241
Qy 241 IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 242 IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 301
Qy 301 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 302 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG 361
Qy 361 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 362 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA 421
Qy 421 ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 422 ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV 481
Qy 481 VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 482 VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS 541
Qy 541 GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 542 GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 601
Qy 601 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 602 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR 661
Qy 661 LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 662 LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 721
Qy 721 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 722 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM 781
Qy 781 KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 782 KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 841
Qy 841 VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 842 VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 901
Qy 901 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 902 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK 961
Qy 961 LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 962 LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM 1021
Qy 1021 IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1022 IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA 1081
Qy 1081 TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1082 TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY 1141
Qy 1141 SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1142 SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 1201
Qy 1201 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1202 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 1261
Qy 1261 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1262 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP 1321
Qy 1321 AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 1367
|||||||||||||||||||||||||||||||||||||||||||||||
Db 1322 AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 1368
Regarding claim 3, Gao et al. describes an gRNA having 100% sequence identity to instant SEQ ID NO: 4, as shown below.
RESULT 3
BGM51116
ID BGM51116 standard; DNA; 487 BP.
AC BGM51116;
DT 22-AUG-2019 (first entry)
DE Wheat U6 promoter target sequence (pTaU6-esgRNA).
KW CRISPR-Cas9 system; U6 gene; crop improvement; ds; genome editing; herbicide resistance; plant; plant breeding; transgenic plant.
OS Triticum aestivum.
OS Synthetic.
CC PN WO2019120283-A1.
CC PD 27-JUN-2019.
CC PF 21-DEC-2018; 2018WO-CN122640.
PR 21-DEC-2017; 2017CN-11393160.
PR 28-APR-2018; 2018CN-10402244.
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
CC PI Gao C, Li C, Zong Y, Wang Y;
DR WPI; 2019-53605Q/53.
CC PT System useful for base editing target sequence in plant genome, comprises
base-editing fusion protein, and guide RNA, and expression construct comprising nucleotide sequence encoding base-editing fusion protein, and guide RNA.
CC PS Example 5; Fig 7; 60pp; English.
SQ Sequence 487 BP; 116 A; 110 C; 125 G; 136 T; 0 U; 0 Other;
Query Match 100.0%; Score 106; Length 487; Best Local Similarity 100.0%;
Matches 106; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 382 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC 441
Qy 61 TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTTTTTTATGTCT 106
||||||||||||||||||||||||||||||||||||||||||||||
Db 442 TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTTTTTTATGTCT 487
Regarding claims 21-23, Gao et al. describes a tRNA adenine deaminase of E coli (p.12, line 11-14) and a TadA DNA-dependent adenine deaminase (SEQ ID NO: 2) (page 13, line 14-16) (as recited in claims 21) comprising 100% sequence identity to instant SEQ ID NO: 33 (as recited in claim 40), as shown below.
RESULT 1
BGM50952
ID BGM50952 standard; protein; 166 AA.
AC BGM50952;
DT 22-AUG-2019 (first entry)
DE Escherichia coli tRNA adenine deaminase (TadA) variant, SEQ ID 2.
KW CRISPR-Cas9 system; DNA-dependent adenine deaminase; TadA protein;
KW crop improvement; genome editing; herbicide resistance; mutein;
KW plant breeding; tRNA adenine deaminase; transgenic plant.
OS Escherichia coli.
CC PN WO2019120283-A1.
CC PD 27-JUN-2019.
CC PF 21-DEC-2018; 2018WO-CN122640.
PR 21-DEC-2017; 2017CN-11393160.
PR 28-APR-2018; 2018CN-10402244.
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
CC PI Gao C, Li C, Zong Y, Wang Y;
DR WPI; 2019-53605Q/53.
CC PT System useful for base editing target sequence in plant genome, comprises
CC PT base-editing fusion protein, and guide RNA, and expression construct
CC PT comprising nucleotide sequence encoding base-editing fusion protein, and
CC PT guide RNA.
CC PS Claim 3; SEQ ID NO 2; 60pp; English.
SQ Sequence 166 AA;
Query Match 100.0%; Score 869; Length 166; Best Local Similarity 100.0%;
Matches 166; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIM 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIM 60
Qy 61 ALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 ALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVL 120
Qy 121 HYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD 166
||||||||||||||||||||||||||||||||||||||||||||||
Db 121 HYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD 166
All the proteins including the specific fusion proteins needed for base editing for editing genomic DNA need to be present in the nucleus in the cell. Gao et al. describes Nuclear Localization Sequence (NLS) (page 15, line 1-3), as recited in claim 26. It teaches one or more NLSs in the base-editing fusion proteins that are sufficient to drive the base-editing fusion protein into the nucleus of a plant cell to achieve base editing (page 15, line 3-6).
Gao et al. describes an adenine base editing system comprising SEQ ID NO: 24, which is suitable for plant cell editing (page 27, line 10-18), besides a DNA dependent adenine deaminase (ABE) from E. coli (page 12, line 11-14), as recited in claims 22-23. SEQ ID NO: 24 of Gao et al. is codon optimized for plant expression and having 100% sequence identity to instant SEQ ID NO: 32 (as recited in claim 41), as shown below.
RESULT 31
BGM50974
ID BGM50974 standard; protein; 809 AA.
AC BGM50974;
DT 22-AUG-2019 (first entry)
DE E. coli TadA-Cas9 fusion protein (SanCas9-ABE-1), SEQ ID 24.
KW CRISPR associated 9; CRISPR-Cas9 system; Cas9 protein;
KW DNA-dependent adenine deaminase; TadA protein; chimeric protein;
KW crop improvement; fusion protein; genome editing; herbicide resistance;
KW plant breeding; tRNA adenine deaminase; transgenic plant.
OS Escherichia coli.
CC PN WO2019120283-A1.
CC PD 27-JUN-2019.
CC PF 21-DEC-2018; 2018WO-CN122640.
PR 21-DEC-2017; 2017CN-11393160.
PR 28-APR-2018; 2018CN-10402244.
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
CC PI Gao C, Li C, Zong Y, Wang Y;
DR WPI; 2019-53605Q/53.
DR N-PSDB; BGM50966.
SQ Sequence 809 AA;
Query Match 100.0%; Score 872; Length 809; Best Local Similarity 100.0%;
Matches 166; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIM 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 2 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIM 61
Qy 61 ALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 62 ALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVL 121
Qy 121 HHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 166
||||||||||||||||||||||||||||||||||||||||||||||
Db 122 HHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 167
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use Cas9 nickase, gRNA(s), and a nucleotide base (adenine or cytosine) editor or a deamination domain from such a nucleotide base (adenine or cytosine) editor to edit target gene(s)/allele(s) in a plant, as described by Brezgin et al. Designing gRNA(s) for specific purpose is a well-known standard process in the art that depends on specific target sequence. Using a specific Cas9 nickase (as recited in claim 38), gRNA(s) (as recited in claim 3), and a DNA-dependent adenine deaminase (as recited in claim 40), as described by Gao et al., having specific structures/sequences is an experimental design choice of an ordinarily skilled artisan without having any realistic possibility of changing the outcome.
Before the effective filing date of the invention, one ordinarily skilled in the art would have been motivated to use a specific Cas9 nickase, gRNA, and a DNA-dependent adenine deaminase to edit target gene(s)/allele(s) in a commercially important plant with high efficiency, as described by Gao et al.
Regarding claims 23-25, All the fused proteins (DNA or RNA dependent aptamer specific binding proteins fused with deaminase protein/domain) acts in trans in a cell. It is an experimental design choice of an ordinarily skilled artisan to make fusion proteins by fusing the aptamer-specific binding proteins (e.g. MCP) with either N-terminal or the C-terminal (as recited in claims 23-24) of an effector protein like adenine deaminase (TadA) or the corresponding adenine deamination domain (as recited in claim 24) using a linker protein. C-terminal deamination domain of TadA proteins including in E coli (as recited in claim 23) (Delannoy et al.; p. 2062, left column, para 3, line 7; page 2069, right column, para 3, line 5-7) and/or in plants (Delannoy et al.; abstract, line 6-8) are known in the art since long. Using a linker (as recited in claims 23 and 25) while making fusion protein(s) by fusing two or more proteins and/or domains is a standard practice in the art, because it offers several advantages such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles (Chen et al.; abstract, last 5 lines) and also as described by Brezgin et al. (p.3, para 6, line 3-5; p.4, para 4, line 3-5).
It is "the normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine” the exact experimental design that works more efficiently in a specific experiment, unless there is evidence indicating a specific experimental design used by the Applicant is critical. Such “critical” feature(s) need(s) to be described by the instant description. See MPEP § 2144.05.
Claim 16-18 are rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above, and further in view of Wang et al. (1) (CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae, 2018, Appl. Environ. Microbiol., 84:e01834-18) and in evidence of Chen et al. (Fusion Protein Linkers: Property, Design and Functionality, 2013, Adv Drug Deliv Rev. 2013 October 15; 65:1357–1369).
Brezgin et al. describes a multiplex genome editing system in various organisms including plants using CRISPR-Cas9 technique comprising nickase nCas9 (nCas9), scaffold RNA (scRNA) containing aptamer sequence(s) (e.g. MS2), aptamer-specific binding proteins (e.g. MCP that binds to MS2 sequence), effector proteins (e.g. cytidine/cytosine deaminases like APOBEC1), as discussed above. Brezgin et al. also describes aptamer-specific binding proteins (e.g. MCP) fused to effector proteins including transcriptional activators (page 6, para 4; page 7, Fig. 2) resulting in fusion proteins (of aptamer-specific binding proteins and effector proteins). Effectors proteins can be a deaminase (either for adenine or cytosine). The fusion proteins are made by fusing the aptamer-specific binding proteins with the N-terminal of the effector protein like cytosine deaminase or cytosine deamination domain (as recited in claim 17), as discussed above. Brezgin et al. also describes use of peptide linkers (as recited in claim 18) to further enhance efficacy of the fusion proteins (page 3, para 6, line 3-5; page 4, para 4, line 3-5).
However, Brezgin et al. does not teach any protein having 100% sequence identity to either SEQ ID NO: 26 (which has been rejoined for compact prosecution) or SEQ ID NO: 29 (the elected species).
Wang et al.(1) describes precise and efficient genome editing in industrial microorganism Klebsiella pneumoniae using cytidine deaminase APOBEC1 and a Cas9 nickase (title; abstract, line 14-15). It also describes a rat APOBEC1 (rAPOBEC1) (page 7, para 2, line 11) (GenBank Accession No. AYA60272) cloned in pBECKP-km vector (page 13, last para, line 3; Fig. 3), which is having 100% sequence identity to instant SEQ ID NO: 26, as shown below.
Title: US-17-909-309A-26
Perfect score: 1275
Sequence: 1 SSETGPVAVDPTLRRRIEPH..........LQSCHYQRLPPHILWATGLK 228
Searched: 1 seqs, 1154 residues
Total number of hits satisfying chosen parameters: 1
Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 1 summaries
Database : AASEQ2_10222025_130526.pep:*
RESULT 1
AASEQ2_10222025_130526
Query Match 100.0%; Score 1275; DB 1; Length 1154; Best Local Similarity 100.0%;
Matches 228; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKH 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 12 SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKH 71
Qy 61 VEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYH 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 72 VEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYH 131
Qy 121 HADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLE 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 132 HADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLE 191
Qy 181 LYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 228
||||||||||||||||||||||||||||||||||||||||||||||||
Db 192 LYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 239
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use a functional equivalent of cytidine deaminase APOBEC1 including the rAPOBEC1, as described by Wang et al.(1), with a realistic goal to edit a genome by precisely converting specific C to T in a gene. Using any specific cytidine deaminase which is functionally equivalent to instant SEQ ID NO: 26 or SEQ ID NO: 29 is an experimental design choice of an ordinarily skilled artisan which would not have expected to change the outcome. Similarly, it also an experimental design choice of an ordinarily skilled artisan to make fusion proteins by fusing the aptamer-specific binding proteins (e.g. MCP) with the N-terminal of the effector protein like cytosine deaminase or cytosine deamination domain (as recited in claim 17) using a linker protein (as recited in claim 18), as discussed above. Using a linker to make fusion protein(s) by fusing two or more proteins/domains is a standard practice in the art that offers several advantages such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles, as described Chen et al. (abstract, last 5 lines) and by Brezgin et al. (p.3, para 6, line 3-5; p.4, para 4, line 3-5).
Before the effective filing date, an ordinarily skilled artisan would have been motivated to use any specific cytidine deaminase rAPOBEC1 fused with an aptamer-specific binding protein using a linker with a realistic goal to edit a genome by precisely converting specific C to T.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above, and further in view of Grunewald et al. (WO2018218206-A1).
Claim 19 depends from claim 1 and is drawn to the first fusion protein comprising an uracil DNA glycosylase inhibitor (UGI), for example, the UGI comprises the amino acid sequence shown in SEQ ID NO: 31.
Brezgin et al. describes a multiplex genome editing system in various organisms including plants using CRISPR-Cas9 technique comprising nickase nCas9 (nCas9), scaffold RNA (scRNA) containing aptamer sequence(s) (e.g. MS2), aptamer-specific binding proteins (e.g. MCP that binds to MS2 sequence), effector proteins, as discussed above. Brezgin et al. teaches that efficacy of cytosine base editors is fairly low due to repair of edited nucleotides by endogenous DNA repair systems like the UNG factor. Improved efficacy of base editing is shown for nickase nCas9 (nCas9) proteins linked to base-editing factors and co-expressed with UGI, an UNG inhibitor. Blocking UNG by UGI transiently impairs DNA repair so that deaminated nucleotides are not corrected.
However, Brezgin et al. does not explicitly describe an uracil DNA glycosylase inhibitor (UGI) comprising of the amino acid sequence shown in SEQ ID NO: 31.
Grunewald et al. describes a Base Editing (BE) technology which is based on the fusion of a DNA binding domain (e.g. catalytically inactive dCas9, nCas9, or nickase) to a cytidine or adenine deaminase and a uracil glycosylase inhibitor (UGI) (page 1, line 14-16). The system comprises: (i) a first fusion protein comprising a programmable DNA binding domain, preferably a transcription-activator-like effector (TALE) or zinc finger array (ZF), fused to a deaminase enzyme, or an active portion thereof, optionally with a linker therebetween, or (ii) a fusion protein comprising a DNA binding domain of Cas9 protein that lacks nuclease activity or is a nickase, but can interact with a guide RNA and target DNA, fused to a Uracil glycosylase inhibitor (UGI), optionally using a linker (abstract). The method also describes targeted deamination of one or more selected cytosines in a nucleic acid (abstract). It describes a UGI protein sequence (SEQ ID NO: 7; Uniprot: P14739) (page 15, line 18-22) having 100% identity to instant SEQ ID NO: 31, as shown below.
BFW14151
ID BFW14151 standard; protein; 83 AA.
AC BFW14151;
DT 24-JAN-2019 (first entry)
DE Bacteriophage uracil glycosylase inhibitor polypeptide, SEQ ID 7.
KW CRISPR-Cas system; CRISPR-Cas9 system; Deamination; UGI protein; Uracil glycosylase inhibitor; genome editing; recombinant protein.
OS unidentified phage.
PN WO2018218206-A1.
CC PD 29-NOV-2018.
CC PF 25-MAY-2018; 2018WO-US034742.
PR 25-MAY-2017; 2017US-0511296P.
PR 04-AUG-2017; 2017US-0541544P.
PR 26-JAN-2018; 2018US-0622676P.
CC PA (GEHO ) GEN HOSPITAL CORP.
CC PA (GRUN/) GRUNEWALD J.
CC PI Grunewald J, Joung JK, Angstman J, Gehrke JM;
DR WPI; 2018-94915V/82.
DR UNIPROT; P14739.
ALIGNMENT:
Query Match 100.0%; Score 418; Length 83; Best Local Similarity 100.0%;
Matches 83; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD 60
Qy 61 APEYKPWALVIQDSNGENKIKML 83
|||||||||||||||||||||||
Db 61 APEYKPWALVIQDSNGENKIKML 83
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use a UGI polypeptide, as taught by Grunewald et al., to improve efficacy of base editing by expressing nickase nCas9 (nCas9) proteins linked to base-editing factors like cytosine deaminase and co-expressed with UGI, as described by Brezgin et al.
Before the effective filing date of the invention, one with ordinarily skilled would have been motivated to use an UGI polypeptide (Uniprot: P14739) to improve efficacy of base editing by expressing a nickase nCas9 (nCas9) protein linked to base-editing factors like cytosine deaminase and co-expressed with UGI.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above, and further in view of Wang et al. (2) (A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis, 2020, aBIOTECH (2020) 1:6–14; Published online on 20 Nov. 2019).
Claim 27 depends from claim 1 and is drawn to the CRISPR nickase, the first fusion protein, and/or the second fusion protein interlinked by a "self-cleavage" peptide.
Brezgin et al. describes a multiplex genome editing system in various organisms including plants using CRISPR-Cas9 technique comprising nickase nCas9 (nCas9), scaffold RNA (scRNA) containing aptamer sequence(s) (e.g. MS2), aptamer-specific binding proteins (e.g. MCP that binds to MS2 sequence), effector proteins, and various fusion proteins, as discussed above.
However, Brezgin et al. does not describe any fusion protein containing a “self-cleavage” peptide.
Wang et al.(2) describes a novel CRISPR/Cas9 system that can generate Cas9-free multiplex mutants efficiently in plants. The system comprises the “most efficient self-cleaving peptide”, 2A, fused to Cas9 and GFP, and then the fused protein Cas9-P2A-GFP is used to generate Cas9-free multiplex mutants efficiently in plants (abstract).
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use a polynucleotide sequence encoding a “self-cleaving peptide”, as described by Wang et al. (2), to interlink Cas nickase and any other fusion protein(s) (as described by Brezgin et al.) with a realistic goal to produce transgene-free genome edited plants, as described by Wang et al.
Before the effective filing date, one with ordinary skill in the art would have been motivated to use a polynucleotide sequence encoding a “self-cleaving peptide” to interlink Cas nickase and any other protein(s) including any fusion protein(s), with the realistic goal to produce transgene-free genome edited plants.
Response to Applicant’s Arguments: The Applicant’s arguments against rejections under 35 U.S.C. §103 boils down to, “Each of the rejections are based on Brezgin as the primary reference, with each of the secondary references asserted to teach or suggest one or more elements of the dependent claims…. Brezgin fails to support a prima facie case of anticipation of instant claim 1” (response, p.15, para 3) and “Brezgin in combination with any or all of Gao, Liu, McConnell, Zalatan, Wang 2018, Grunewald, and/or Wang 2020 fails to support a rejection of instant claim 1 under 35 U.S.C. §103” (response, p.15, last para line 1-3).
The Examiner disagrees as Brezgin et al. fulfils all the claim limitations of instant claim 1, as discussed above.
Conclusion
No claim is allowed.
Claims 10-11 are objected to as being dependent upon a rejected base claim.
Applicant's amendment necessitated the new grounds 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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J.C.
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
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