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 1-5 are pending and examined here. This is a 3rd non-final action.
Priority
The priority to RU2018141534, filed on 11/26/2018, via its PCT/RU2019/050230, filed on 11/26/2019 is acknowledged.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
The date of 11/26/2019 will be used for the purposes of prior art search.
All claims enjoy the priority to 11/26/2019, the date of PCT filing since a certified translation of foreign application is not provided.
Nucleotide and/or Amino Acid Sequence Disclosures
The text of the sections of Nucleotide/Amino Acid sequence disclosure is noted in prior Office Action.
The objection regarding nucleotide disclosure of Fig. 1, 6, and 9 is withdrawn, however objection regarding Fig. 3 is maintained. The fig. 3 has 4 sequences, but only two sequence identifiers are disclosed. Illustrative region of target DNA can be altered or will require sequence identifiers, since the nucleotide sequences are greater than 9 nt.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Pg. 6 provides link to NCBI: http:. . . .
Claim Objections
Claim 4 is objected to because of the following informalities: the required elements need better organization; currently, it reads as limitation of a), or b), and c), or d), although it is interpreted as (a or b) and (c or d), it can be read as a, or (b, and c), or d. The following suggested phrasing would be better:
an effective amount of a) a protein . . . or a nucleic acid . . ., and b) a guide RNA . . . or a DNA sequence.
Additionally, the use of “which” in line 11 of cl. 4 grammatically refers to the “organism” but it is understood that “which” phrase is referring to the duplex formation that is directly adjacent to the PAM sequence. But a skilled artisan also understands that a single guide RNA (gRNA) is designed to bind to a complementary strand of a genome that is nearby a PAM sequence, since a canonical Cas enzyme will not cut the DNA without a PAM sequence (and is supported by the specification by their examples (pg. 9-12 or pg. 12, 3rd full paragraph) and gRNA also recruits the Cas enzyme.
Thus, claim 4 would be better as “an effective amount of a) a protein comprising the amino acid sequence of SEQ ID NO: 1 or a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1, and b) a guide RNA that is complementary to the genomic DNA sequence comprising 5’-NN(G/A)NA(C/T)N-3’ or a DNA sequence encoding said guide RNA.”
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Scope of Enablement Rejection:
Claims 1-5 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 of forming a double-strand break in a DNA molecule, the method comprising introducing into a bacterial cell comprising the DNA molecule: a protein comprising the amino acid sequence of SEQ ID NO: 1 or a nucleic acid encoding the protein of SEQ ID NO: 1 and a guide RNA, wherein the DNA molecule comprises the PAM sequence 5’-NN(G/A)NA(C/T)N-3’, but does not reasonably provide enablement for “A method of forming a double-strand break in a DNA molecule, where the double-strand break is 0 to 25 nucleotides before the nucleotide sequence 5’-NN(G/A)NA(C/T)N-3’ in the DNA molecule, the method comprising introducing into a cell comprising the DNA molecule: a protein comprising the amino acid sequence of SEQ ID NO: 1, or a protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 and differs from SEQ ID NO: 1 only in non-conserved amino acid residues” (cl. 1-3) nor for “a method of generating a double-strand break in a genomic DNA sequence of a unicellular or multicellular organism directly adjacent to the sequence 5’-NN(G/A)NA(C/T)N-3’, comprising introducing into at least one cell of said organism, an effective amount of a) a protein comprising the amino acid sequence of SEQ ID NO: 1, or b) a nucleic acid encoding the protein comprising the amino acid sequence of SEQ ID NO: 1, and c) guide RNA forms a duplex with the nucleotide sequence of the genomic DNA sequence of the unicellular or multicellular organism, which is directly adjacent to the nucleotide sequence 5’-NN(G/A)NA(C/T)N-3’ and interacts with said protein following the formation of the duplex, or d) a DNA sequence encoding said guide RNA. . . “ (cl.4-5). The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
When considering the scope of enablement, the Wands factors need to be reviewed, which poses whether the experimentation needed to practice the invention is undue or unreasonable. Determining undue experimentation requires analysis of, but 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 the instant case: Claims 1 and 4 are noted above.
The breadth of the claim: For claim 1, the breadth of claim encompasses a method of forming a double-strand break in a DNA molecule following a PAM sequence (the recited 5’NN(G/A)NA(C/T)N-3’ will also be called the “PAM sequence” for protospacer adjusted motif sequence) by introducing in a cell a protein comprising SEQ ID NO: 1 or one with claimed 95% identity to SEQ ID NO: 1 without requiring guide RNA. For claim 4, the method is directed to forming a double-strand break directly adjacent to the recited PAM sequence in a genomic DNA sequence of any unicellular or multicellular organism by introducing to any one cell of said organism an effective amount of a) protein of SEQ ID NO: 1 or a nucleic acid encoding the protein of SEQ ID NO: 1 and b) gRNA or DNA sequence encoding gRNA, where the interaction between said protein with gRNA and the PAM sequence results in the formation of a double-strand break in the genomic DNA sequence adjacent to the PAM sequence.
The nature of the invention: The nature of the invention is a method of forming a double-strand break in a DNA molecule in a cell or an organism either with a protein comprising claimed SEQ ID NO: 1 (or its claimed variants) along with a guide RNA or nucleic acid encoding the protein of claimed SEQ ID NO: 1 along with gRNA (or its claimed variants).
The state of the prior art:
Uncertainty of double-strand break without gRNA in claims 1-3: Jian and Doudna (2017, Annual Review of Biophysics, 26, 505-529) in their CRISPR review article indicate that “[t]arget recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9–DNA interactions, and associated conformational changes” in a dual RNA-guided DNA endonuclease Cas9 cleavage (abstract). It is understood that other Cas isoforms, i.e. instead of Cas9 specifically, will also cleave (see pg. 508 in CRISPR-Cas9 Biology section). Thus there is uncertainty whether a Cas enzyme by itself will result in formation of a double-stranded break in a DNA molecule.
Uncertainty of genome modification in a non-bacterial cell (cl. 1-5): In the Inventors post-filing article (Fedorova et al., 2020, Nucleic Acids Research, 48, 12297-12309, “Fedorova”, in IDS of 10/12/2022, cite 5), the inventor disclose that “[n]o genome modification was detected in cells transfected with DfCas9 (data not shown)” (pg. 12304) when a DfCas9, which was codon optimized, was cloned into a plasmid vector under the control of a CMV promoter and was transfected into HEK293T cells and production of recombinant Cas9 proteins was confirmed (pg. 12304). Chen et al. (2017, Nat Commun., 8:14958, pg. 1-12) highlight that “many CRISPR-Cas were found inactive in human cells even though they were active in bacteria or on purified DNA substrates . . . .currently there is lack of methodology to utilize inactive CRISPR-Cas systems in mammalian genome-editing applications” (pg. 2). Thus, it is uncertain that expression of DfCas9 or its claimed variant will result in formation of double-strand break in a DNA molecule in any cell or in any unicellular or multicellular organism, especially in a non-bacterial cell, since as Fedorova discloses despite expression of DfCas9 in HEK293T cells the authors did not observe genome modification, which initially requires double-strand DNA break in the genome for genome modification.
Uncertainty regarding double-strand break 0 to 25 nt. before the PAM sequence (regarding cl. 1-5: Claim 4 is directed to a double-strand break “directly adjacent” to the PAM sequence. The “directly adjacent” language in light of the specification pg. 6 is understood as a break in a target sequence made at a distance of 0 to 25 nt. before the PAM sequence. Thus, it has a similar interpretation as cl. 1 regarding the location of double strand break relative to the PAM sequence. Here, there is also uncertainty regarding the distance between a break in genomic DNA and the nucleotide sequence 5’-NN(G/A)NA(C/T)N (aka the PAM sequence) in the genomic DNA. Here the claims are directed to the double-strand break is 0 to 25 nt., thus can be interpreted that the break can form anywhere between 0 to 25 nt. from the recited PAM sequence. However, as understood in the art, DNA break following Cas9 cleavage is generated at a specific single site, i.e. some specific distance from the PAM sequence on each strand (see Fig. 3, pg. 510 of Jiang and Doudna). The Fig. 3 illustration indicates the two domains (HNH and RuvC domains) of Cas9 each cutting a single site within each strand of a genomic DNA 3 nt. from the PAM sequence (also pg. 519). Ran et al. (2015, Nature, 520, 186-191) in their Extended Fig. 2 comparing Cas9 orthologues cleavage pattern in vitro indicate that, first, “most Cas9 enzyme cleaves stereotypically at 3bp upstream of PAM” and, second, the stacked bar graph data indicate that the minimal distance is 2 nt. between the PAM sequence and the break in the DNA (see below). None of the Cas9 orthologues examined had a 0 distance between the DNA cleavage and the PAM sequence.
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540
554
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Thus, there is uncertainty whether there is variability in the length (ranges of 0-1 nt. and from 6 nt. to 25 nt. distance between the break and the PAM sequence) of a double-strand break in genomic DNA comprising the PAM sequence in bacterial cell.
Uncertainty of claimed SEQ ID NO: 1 variants forming/generating double-strand break in any non-bacterial cell: Claim 1 and its dependent claims 2-3 are directed to “an amino acid sequence of SEQ ID NO: 1 and differs from SEQ ID NO: 1 only in non-conserved amino acid residues.” The issue, as noted above, is that the post-filing reference indicating the lack of genome modification within a mammalian cell, is in addition to lack of enabled species that differ from SEQ ID NO: 1 in non-conserved amino acid residues that forms a double-strand break in any cell.
The level of one of ordinary skill: The level of one of ordinary skill is high.
The level of predictability in the art: Although there are many details unknown of CRISPR system double-stranded break, as Jiang and Doudna point out that guide RNA (gRNA) is required for a Cas9 (or an equivalent Cas enzyme) cleavage. Further, as Chen discloses despite having general understanding of Streptococcus pyogenes derived Cas9 (SpCas9) and a few other CRISPR-Cas systems from other bacteria (Staphylococcus aureus, SaCas9) to edit within mammalian cells (pg. 2), there are “many CRISPR-Cas systems that had been explored for mammalian gene editing were found inactive in human cells” (pg. 2). Further as noted above Fedorova discloses that despite expression of DfCas9 in human cell line, the authors did not observe genome editing. Thus, the art is unpredictable that a DfCas9 or its variants would cause double-strand break in any cell or in any unicellular or multicellular organism (regarding instant cl. 1-5).
The amount of direction provided by the inventor: Regarding the lack of gRNA for cl. 1-3; Pg. 2 of specification notes that type II CRISPR-Cas9 system “requires the formation of an effector complex consisting only of one Cas9 protein and two short RNAs as follows: crRNA and tracer RNA (tracrRNA). The tracer RNA complementarily pairs with a crRNA region, originating from CRISPR repeat, to form a secondary structure necessary for the binding of guide RNAs to the Cas effector.” It should be clarified that synthetic single guide RNA (sgRNA or gRNA) also encompasses a single entity sequence that mimics the combination of crRNA (CRISPR RNA) and tracrRNA (pronounced “tracer RNA” and is transactivating CRISPR RNA) sequences, see Jiang and Doudna (abstract). Further the specification highlights “[t]o develop a system for cutting DNA in vivo and in vitro, it is necessary to obtain all components that are part of the effector DfCas9 complex, as follows: guide RNAs and DfCas9 nuclease” (pg. 10). Thus, the Inventor recognizes that both a gRNA and a Cas enzyme is required, and there is uncertainty whether the protein encoded by claimed SEQ ID NO: 1 by itself will form a double-stranded break.
The existence of working examples:
a. Uncertainty of double-strand break without sgRNA: There are no working examples demonstrating the formation of dsDNA break by dfCas9 and DNA template without sgRNA. However, the specification provides in vitro examples with dfCas9 and gRNAs (or tracrRNA and crRNA). The specification provides a mixture containing sgRNA, crRNA, tracrRNA, DfCas9, and/or target DNA in a test tube to demonstrate cleavage in target DNA (pg. 11, Fig. 4 illustrating DNA breaks, see also Example 3, pg. 14, Fig. 8 below).
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The column labeled “control (without RNA),” is enzyme by itself and no gRNA (or crRNA and tracrRNA). Thus, Fig. 8 clearly indicates both gRNA and a Cas9 is required. Thus, it would be uncertain if the Cas9 by itself will result in formation of dsDNA break.
b. Uncertainty of genomic modification in a non-bacterial cell: The specification provides transformation of E. coli bacterial cells with DfCas9 construct in determining gRNA and PAM sequence (pg. 9-10). However, as noted above, the inventors own publication noting the lack of genomic modification in human cells and Chen indicating that many CRISPR-Cas systems that had been explored for mammalian gene editing were found inactive in human cells suggest that it is uncertain whether a double-strand break is formed/generated in mammalian cell, in non-bacterial unicellular organism or multicellular organism by protein of SEQ ID NO: 1 (cl. 1-5).
c. Uncertainty of double-strand break 0 to 25 nt. before the PAM sequence: Although the specification provides ample evidence of an in vitro cleavage in bacterial cell or in test-tube, the specification does not provide for demonstration of exact distance of cleavage from the PAM sequence, it speculates based on the length of gel products. Thus, there is uncertainty whether there is variability in the length (i.e. any nt. position 0 to 25) of formation of a double-strand break in genomic DNA comprising the PAM sequence in bacterial cell.
d. Uncertainty of claimed SEQ ID NO: 1 variants forming/generating double-strand break in any non-bacterial cell: Here, the specification besides providing an alignment between SaCas9 (Staphylococcus aureus), CjCas9 (Campylobacter jejuni) and DfCas9 with the non-conserved regions underlined (see Fig. 9) does not provide for factors, i.e. the database used, algorithmic cut-offs, that determine the sequence that was non-conserved (i.e. underlined). The specification does not demonstrate various species that are 95% identical to the SEQ ID NO: 1 that differs from SEQ ID NO: 1 only in non-conserved amino-acid residues that form double-strand break in any cell, including bacterial cell. As Chen discloses that many CRISPR-Cas systems that had been explored for mammalian gene editing were found inactive in human cells, there is uncertainty whether the claimed variant will be form double-strand break in any non-bacterial cell (cl. 1-3).
The quantity of experimentation: Thus, due to the uncertainty of whether DfCas9 without gRNA (tracrRNA and crRNA) will result in formation of dsDNA break in a non-bacterial cell, there is a need for undue experimentation, which will most likely by unsuccessful. Further, there is uncertainty even a combination of DfCas9 or its claimed variant and gRNA (tracrRNA or crRNA) will form a double-strand break in a DNA molecule comprising the PAM sequence in any non-bacterial cell. There is also uncertainty regarding the double-strand is any variable length from 0 to 25 nt. nucleotides before the PAM sequence. Thus, cl. 1-3 are rejected, as none of the dependent claims provide for combination of SEQ ID NO: 1 protein and gRNA together nor of forming a double-strand break in any non-bacterial cell. Further, there is uncertainty whether protein of (or nucleic acid encoding) SEQ ID NO: 1 with sgRNA will generate a double-strand break in a genomic DNA sequence comprising the PAM sequence of a non-bacterial unicellular organism or multicellular organism. Thus, cl. 4-5 are rejected since undue experimentation would be required.
112(b)
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 1-3 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 term “non-conserved,” line 8 in claim 1 is a relative term which renders the claim indefinite. The term “non-conserved,” in terms of “a protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 and differs from SEQ ID NO: 1 only in non-conserved amino acid residues” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The non-conserved sequence is not clearly defined, as to what is the “metes and bounds” of a non-conserved sequence and what is a conserved sequence, and not just based on the noted Cas9s, since there are other Cas9s, the conserved or non-conserved site also depend on the database and algorithm selected to determine “conserved” vs. “non-conserved.” Although Fig. 9 is illustrative, which provides amino acid alignment of Cas9 protein sequences from Staphylococcus aureus, Campylobacter jejuni, and DfCas9 and the non-conserved regions underlined (pg. 5-6), the Figure does not provide sufficient guidance to a skilled artisan as to how the “underlined” sequence that is “non-conserved” was determined and therefore difficult to extrapolate to other Cas9s. Cl. 2-3 do not overcome the indefiniteness.
Claim Rejections - 35 USC § 103
Applicant’s arguments, see pg. 5-8, filed 10/09/2025, with respect to the rejection of claims 1-5 under 35 USC 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made, as noted below.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-5 are rejected under 35 U.S.C. 103 as being unpatentable over Doudna et al. (US20140068797, pub. 03/06/2014, “Doudna”) and GenBank® Acc: SDW73882.1, (www.ncbi.nlm.nih.gov/protein/SDW73882.1/, of record).
To be consistent with the enablement rejection, the art rejections of cl. 1-5 are only in bacterial cell, as all the claims encompass bacterial cell and for cl. 4-5, the double-strand break “directly adjacent” to the claimed PAM sequence is interpreted, in light of the specification, as between 0 to 25 nt. (pg. 6).
Regarding instant cl. 1, 3, 4, NCBI site discloses a CRISPR-associated endonuclease, Csn1 family (Allgaiera indica), with accession number SDW73882, which was available as of 2/2/2017 and is of 1079 a.a., which is 100% identical to instant SEQ ID NO: 1 (see alignment below, relevant to instant cl. 1, 3, 4). Csn1 is also known as Cas9 (see Doudna, par. 620).
NCBI’s SDW73882 does not disclose the elements of cl. 1-5.
Doudna discloses that Type II CRISPR-Cas systems are widespread in bacteria and disclose screening publicly available genomes for sequence homologous to already annotated Cas9 proteins to identified 235 Cas9 orthologues, conducting sequence alignment, conducting a deeper sequence analysis of subclustering of the sequences (par. 707-710). Doudna discloses that following alignment of Cas9 orthologues, the comparative analysis “revealed high diversities in amino acid composition and protein size” and “share only a few identical amino acids” but share a “same domain architecture with a central HNH endonuclease domain and splitted RuvC/RNaseH domain” and range from 984 to 1624 amino acids (par. 710). Doudna purified a bacterial produced Cas9 and performed cleavage analysis (par. 44, Fig. 12, par. 622). Doudna also screened for PAM sequences by testing a series of dsDNA duplexes containing mutations in the PAM sequences on the complementary or non-complementary strands, or both (Fig. 13A) to identify PAM sequence associated with the Cas9 protein (par. 630). Doudna demonstrated studies in test tube as well as transforming E.coli cells with wild-type Cas9 and tracrRNA and crRNA (par. 607, see Fig. 26, relevant to instant cl. 4). Fig. 26 illustrates results of “transformation assays demonstrating that the GG motif is essential for protospacer plasmid DNA elimination by CRISPR/Cas in bacterial cells” (par. 630, see Fig. 26). Doudna demonstrates Cas9 endonuclease can be programmed with guide RNA (gRNA) engineered as a single transcript to target and cleave any dsDNA sequence of interest (par. 638).
One of the KSR’s rationale for supporting conclusion of obviousness is “obvious to try,” requiring the following three findings: (1) a finding that at the relevant time, there had been a recognized problem or need in the art, which may include a design need or market pressure to solve a problem; (2) a finding that there had been a finite number of identified, predictable potential solutions to the recognized need or problem; (3) a finding that one of ordinary skill in the art could have pursued the known potential solutions with a reasonable expectation of success.
To identify and characterize Cas9 proteins for method of site-specific modifications of a target DNA (par. 21), Doudna discloses conducting characterization of various features of the Cas9 enzyme, including identifying homologous Cas9 through genome sequence analysis and identifying its respective PAM sequence. Therefore, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have performed search through public databases to identify homologous Cas9, including NCBI’s SDW73882, in view of Doudna to have arrived at the claimed invention with a reasonable expectation of success. Here, Doudna teaches a method to conduct a screen through public database to identity homologous Cas9, then to characterize its feature, including identify its PAM sequence, thus a skilled artisan would reasonably expect success in trying to characterize the SDW73882 Cas9’s properties, including its method to form a double-strand break and identifying its PAM sequence through a PAM screen. Thus, cl. 1 is obvious.
Regarding instant cl. 2, Doudna discloses incubating native or restriction digest-linearized plasmid DNA with purified Cas9 protein and tracrRNA:crRNA duplex in a Cas9 plasmid buffer at 37°C for 60 min (par. 597). Doudna further conducted enzyme co-factor analysis of divalent cations, including Mg, Mn, Ca, Zn, see Fig. 18, par. 609). Thus, it would be obvious for a skilled artisan based on Doudna’s characterization of Cas9 enzyme, a skilled artisan would reasonably expect success trying to characterize Cas9 enzyme of SDW73882 by testing various divalent cations, including Mg, Mn, and Ca, as taught by Doudna. Thus, cl. 2 would be prima facie obvious.
Regarding instant cl. 5, Doudna discloses that following a cleavage of double strand breaks, the breaks are repaired by a cell in one of the two ways: one way is homology-directed repair (HDR) by the use of homologous template, the other is non-homologous end joining (NHEJ) repair where there is direct ligation of the break ends to one another (par. 151, 258). The HDR method a donor polynucleotide with homology to the cleaved target DNA sequence is used as a template for repairing cleaved target DNA sequence with introducing of a new nucleic acid material is inserted into a subject cell (par. 258). Doudna discloses that donor polynucleotide may be used to “knock-in” a nucleic acid to induce a 2-fold increase or more in the amount of target modification (par. 260, 297).
Thus a skilled artisan would have been motivated to characterize the NCBI’s SDW73882 Cas9 in view of Doudna, and introduced donor polynucleotide to knock-in a gene in a more efficient manner. Thus, cl. 5 would be prima facie obvious.
Response to Arguments
As noted above, the arguments of Remarks were persuasive and the rejection based on prior references was withdrawn. Although new references have been used, some art is the same, i.e. the GenBank® disclosure.
The Remarks of 10/09/2025 argues that the NCBI GenBank disclosure does not teach the use of the sequence (pg. 5), nor that the enzyme is “active and can modify specific sequences introducing sdDNA breaks upstream of the” claimed PAM sequence (pg. 6), thus “NCBI GenBank fails to disclose any properties of the DfCas9 of SEQ ID NO: 1 and, its activity is only based on sequence similarity” (pg. 6).
The argument is not persuasive, since the sequence of GenBank® Acc: SDW73882.1 is known in the art prior to the effective filing date of instant application, it would be obvious to try to characterize a homologous Cas9 enzyme in a public database either through sequence analysis or search of Cas9/CRISPR enzymes, and then characterizing its cleavage activity, including identifying its PAM sequence, as discussed by Doudna. Doudna discloses a screen to identify the corresponding PAM sequence of a Cas9 through a mutational analysis of PAM sequence. Karvelis et al. (2017, Methods, 121-122, pg. 3-8) discloses various ways PAM sequence can be identified for a Cas9 protein.
Allowable Subject Matter
No claim allowed.
Sequence Alignment:
Csn1, Allgaiera indica, SDW73882.1 (Qy) and instant SEQ ID NO: 1 (Db)
Query Match 100.0%; Score 5665; DB 1; Length 1079;
Best Local Similarity 100.0%;
Matches 1079; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MYRFAFDLGTNSLGWAVYRLTNDTRPASIERTGVRIFPNGRDPQSKESNAAGRRMPRGAR 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MYRFAFDLGTNSLGWAVYRLTNDTRPASIERTGVRIFPNGRDPQSKESNAAGRRMPRGAR 60
Qy 61 RRQDRSLGRRKRLLDDLIGFGLLPSDADARSAVFAANPIEARARAAREQVALEQLGRALW 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 RRQDRSLGRRKRLLDDLIGFGLLPSDADARSAVFAANPIEARARAAREQVALEQLGRALW 120
Qy 121 HMSKHRGFKSNRRADKDADEKGKIAIA SAALLERLRADGHPTYGAFLHARLVRGEGTRIR 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 HMSKHRGFKSNRRADKDADEKGKIAIA SAALLERLRADGHPTYGAFLHARLVRGEGTRIR 180
Qy 181 PAGDGAKLSYEFYPTRALLEAEFDHIWAIQAGFHPSLTEAMHERLRDTIFFQRPLRPVRP 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 PAGDGAKLSYEFYPTRALLEAEFDHIWAIQAGFHPSLTEAMHERLRDTIFFQRPLRPVRP 240
Qy 241 GKCTFFPDQDRLPRWHPAAQEFLILSQLNHLRIVDDQGEQPLDITARDLVARTLMAGTKL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GKCTFFPDQDRLPRWHPAAQEFLILSQLNHLRIVDDQGEQPLDITARDLVARTLMAGTKL 300
Qy 301 SWSGLKKTLKLPSQAEFNLEKGGLKELARNDVAARLLGDTKKPGPLATLWPTLDSATQEE 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 SWSGLKKTLKLPSQAEFNLEKGGLKELARNDVAARLLGDTKKPGPLATLWPTLDSATQEE 360
Qy 361 ILWQISEVADPEELICWLMERLGLSREVAERVEKIPLPDGHLRFCKTATQAIVEKLRCDV 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 ILWQISEVADPEELICWLMERLGLSREVAERVEKIPLPDGHLRFCKTATQAIVEKLRCDV 420
Qy 421 IPYDEAVRRAPLLGGAGLDHSDFQAEEGVDTLPPYNRLPVLQRMIGNGTSDPKDTDLLRY 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 IPYDEAVRRAPLLGGAGLDHSDFQAEEGVDTLPPYNRLPVLQRMIGNGTSDPKDTDLLRY 480
Qy 481 GRITNPTVHIALGQFRRVMNALIAEYGKPAQVVIEATRDMAKSAEELNKIEKTIRDNEKR 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 GRITNPTVHIALGQFRRVMNALIAEYGKPAQVVIEATRDMAKSAEELNKIEKTIRDNEKR 540
Qy 541 NDRWRAELQKAGLLVEGARIGDRFLRMRLWEEIGRGPADRLCPYTGRPIALHQLHSDEIE 600
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Db 541 NDRWRAELQKAGLLVEGARIGDRFLRMRLWEEIGRGPADRLCPYTGRPIALHQLHSDEIE 600
Qy 601 IDHILPFEQTFDDSPANKSLCFRDANRRKGKLSPAAAADRQPDFFDLAAIIGRTKHLPAN 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 IDHILPFEQTFDDSPANKSLCFRDANRRKGKLSPAAAADRQPDFFDLAAIIGRTKHLPAN 660
Qy 661 KAWRFLPGAMEKWEETRGFEDRQLNATGYLARVVRAYTEALFPKTDAEGKRRSHVWVLPG 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 KAWRFLPGAMEKWEETRGFEDRQLNATGYLARVVRAYTEALFPKTDAEGKRRSHVWVLPG 720
Qy 721 RMTAMLRHRWGLNLGDHNRKSRDDHRHHAIDAAVVGVIDRRMVQVLQTHARNLGVERLDR 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 RMTAMLRHRWGLNLGDHNRKSRDDHRHHAIDAAVVGVIDRRMVQVLQTHARNLGVERLDR 780
Qy 781 VLPAPPEPFEGFRDAVLAAVEKVNVSHRAQHGSIDPTDPSQTSGRLHEDTVFGLVRDVPE 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 VLPAPPEPFEGFRDAVLAAVEKVNVSHRAQHGSIDPTDPSQTSGRLHEDTVFGLVRDVPE 840
Qy 841 NQAERTIGNVVVRKPITGLTEKEIGQVRDVKMRLSLQDATSLARDKSLPEAERKKRLPEA 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 NQAERTIGNVVVRKPITGLTEKEIGQVRDVKMRLSLQDATSLARDKSLPEAERKKRLPEA 900
Qy 901 LAKWVQETGHRKLRILKPEAGVRPVHDRDGKPYKWLVPGEISWLDILEAPDGTWFQHATD 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 LAKWVQETGHRKLRILKPEAGVRPVHDRDGKPYKWLVPGEISWLDILEAPDGTWFQHATD 960
Qy 961 IWAANSGGAEPWNVAHPSARFIMRVHKNDTIQVFDWDDRNKCVVEGSNQIKRIVRLEPSA 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 IWAANSGGAEPWNVAHPSARFIMRVHKNDTIQVFDWDDRNKCVVEGSNQIKRIVRLEPSA 1020
Qy 1021 NRMRLVGINDAGKFDERHNEPNDPFRWDLATISKLKLRRARRVRIDELGRVHTIPHGTV 1079
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 NRMRLVGINDAGKFDERHNEPNDPFRWDLATISKLKLRRARRVRIDELGRVHTIPHGTV 1079
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KEYUR A. VYAS whose telephone number is (571)272-0924. The examiner can normally be reached M-F 9am - 4 pm (EST).
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Jennifer Dunston can be reached at 571-272-2916. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KEYUR A VYAS/Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637
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