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 .
Priority
This application is a 371 of PCT/US2022/023136 which claims the benefit of US Provisional Application 63/172,942 with an effective filing date of 09 April 2021 as reflected in the filing receipt mailed on 26 June 2024.
Status of the Claims
Claims 1-17, 19, 20, and 27 are pending.
Claims 4, 7-11, and 13-17 are amended.
Claims 18, 21-26, 28, and 29 are cancelled.
Information Disclosure Statement
The information disclosure statement (IDS) submitted is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Specification
The disclosure is objected to because of the following informalities:
The use of the terms Bless, INDUCE-seq, MinElute, Qiagen, ApeXBio, CRISPRMAX, Opti-MEM, Monarch, Sigma, which appears to reference Sigma-Aldrich, AMPure, Covaris, Dynabeads, Life Technologies, Tween, NEBNext, Thermomixer, NextSeq, CAS, Synthego, IDT, etc. throughout the instant specification, which are trade names or marks used in commerce, has been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Appropriate correction is required.
Claim Objections
Claims 1 and 19 are objected to because of the following informalities:
Claim 1 includes the identification parenthetical “single stranded DNA (ss-DNA)” in step (a). Parentheticals within claim language may lead to a lack of clarity. The claim is interpreted without the parenthetical as “single stranded DNA,,”.
Claim 1 step (a) states “ss-DNA”. Claim 1 step (b) states “ssDNA”. For consistency amongst the claims, the single stranded DNA identification should all be “ss-DNA” or “ssDNA”.
Claim 19 step (g) states “ss-DNA”. Claim 19 step (h) states “ssDNA”. For consistency amongst the claims, the single stranded DNA identification should all be “ss-DNA” or “ssDNA”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 19 and 20 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
If the language of the claim is such that a person of ordinary skill in the art could
not interpret the metes and bounds of the claim so as to understand how to avoid
infringement, a rejection of the claim under 35 USC 112(b) is appropriate, see MPEP
2173.02.
Claim 19 starts with step (g). Claim 19 depends from claims 1 and 15. Claims 1 and 15 end with step (e). Therefore, claim 19 lacks clarity because it is missing step (f). To provide some form of clarity regarding the steps, claim 19 is interpreted to depend from claim 15 and claim 15 is interpreted to depend from claim 6.
Claim 19 depends from claims 1 and 15. Claim 19 step (g) appears to be performed after the claim 6 step (f) of washing and amplification. Therefore, claim 19 lacks clarity as to whether or not the amplified DNA fragment comprising the first binding member of the specific binding pair of step (f) is then contacted in step (g) with an RNA-guide endonuclease without the guide RNA … . Herein, claim 19 is interpreted as an independent claim comprising steps (a)-(e).
Claim 19 step (g) states “contacting an RNA-guided endonuclease without the guide RNA with the target double-stranded DNA inside a control cell, thereby producing ss-DNA at the site to which the guide-RNA binds”. It is unclear within the claims as to the process in which an RNA-guided endonuclease without the guide RNA will bind to the DNA at the site to which the guide-RNA binds. The instant specification appears to not provide guidance regarding the process in which an RNA-guided endonuclease without the guide RNA will bind to the DNA at the site to which the guide-RNA binds. Claim 19 step (g) lacks clarity and is interpreted as “contacting an RNA-guided endonuclease without the guide RNA with the target double-stranded DNA inside a control cell, thereby producing ss-DNA at a binding site
Claim 20 depends from base claim 19 and is included in this rejection as it
does not correct the informalities identified in base claim 19.
Claim Interpretation
Claim 19 depends from claims 1 and 15. Claim 19 step (g) states “contacting an RNA-guided endonuclease without the guide RNA with the target double-stranded DNA inside a control cell, thereby producing ss-DNA at the site to which the guide-RNA binds”. Claim 1 preamble and step (a) state a “method for isolating DNA fragments that contain a binding site for a guide-RNA of an RNA-guided endonuclease, the method comprising: (a) contacting the RNA-guided endonuclease with a target double-stranded DNA, thereby producing single stranded DNA (ss-DNA) at the site to which the guide-RNA binds”. The term “an RNA-guided endonuclease without the guide RNA” in claim 19 appears contradictory to claim 1. It appears the term is included in claim 19 to distinguish between “an RNA-guided endonuclease” in claim 1 and “an RNA-guided endonuclease without the guide RNA” in claim 19. Herein, claim 1 is interpreted to comprise an RNA-guided endonuclease, such as Cas9 guide RNA, and claim 19 is interpreted to comprise an RNA endonuclease, such as Cas9, see instant specification, Pg. 27, Ln. 19-Pg. 30, Ln. 5.
In the Spirit of Compact Prosecution
While the examiner has attempted to identify all objections and clarity issues amongst the claims, applicant is advised that some objections and clarity issues may still remain. Going forward, the examiner respectfully requests applicant to perform a detailed review of the claims regarding clarity, grammar, antecedent basis, word spacing, and spelling issues.
Claim Rejections - 35 USC § 103
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 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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-17, 19, 20, and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al., (“Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ”, Published online 06 April 2020, see Pg. 522, Col. 1, Bottom and PDF Pg. 33 for the online publication date and the URL, Nature Methods, Vol. 17, Pgs. 515-523 and Supplementary PDF Pgs. 10-33, hereinafter Wu) in view of Zhang et al. (WO2020006036, published 02 January 2020, hereinafter Zhang).
Wu is in the known prior art field of “a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N3-kethoxal and guanines in ssDNA”, see Abstract; Pg. 516, Fig. 1.
Regarding instant application claims 1-4, Wu teaches a method of isolating DNA fragments by step 1.) isolating a single strand of a target DNA and contacting a target double stranded RNA polymerase II, such as Pol II, with N3-kethoxal, i.e., kethoxal modified by the chemo selective group N3, in order to obtain/label single-stranded guanines at the site the Pol II binds in the genome, see Pg. 516, Fig. 1 and below,
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, see also, Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting:
Step (b) in instant application claim 1; and,
The specific azido of N3 and the chemo selective group N3-kethoxal in instant application claim 2;
In step 2.) the chemo selective group is linked to a biotin binding member, see above and Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting step (c) in instant application claim 1;
In step 3.) the DNA is fragmented, see above and Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting step (d) in instant application claim 1;
The DNA single-stranded fragments are then enriched through the biotin–streptavidin interaction and subjected to library construction, followed by PCR -amplification and HT-sequencing, see above and Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting:
The method of isolating DNA fragments and step (e) in instant application claim 1;
The specific binding pair of biotin–streptavidin in instant application claim 3; and,
The amplification in instant application claim 4.
Regarding instant application claim 5, Wu teaches the binding pair of biotin–streptavidin beads are present in the amplification because the PCR input is DNA or the beads suspension, see PDF Pgs. 31-32, Low-input KAS-seq, meeting the amplifying of the DNA with the binding pair in instant application claim 5.
Regarding instant application claim 6, Wu teaches washing the biotin–streptavidin beads 5 times with 1×binding and wash buffer prior to the PCR amplification of the beads suspension, see PDF Pgs. 31-32, Low-input KAS-seq, meeting the enrichment of the DNA with the binding pair in instant application claim 6.
Regarding instant application claim 7, Wu teaches isolating the DNA cells, such as with a 5 minute wash away, after N3-kethoxal labeling and before biotinylation, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting the washing before the first binding pair in instant application claim 7.
Regarding instant application claim 8, Wu teaches the target DNA is a genomic DNA, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting the target DNA in instant application claim 8.
Regarding instant application claims 11 and 12, Wu teaches after amplification the DNA fragment with the specific binding site undergoes HT-sequencing “on Illumina Nextseq500 platform with single-end 80-bp mode, aiming to get 30 million reads per library” aka a next generation sequencing method, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting:
The sequencing and the sequencing device in instant application claim 11 and in instant application claim 12.
Regarding instant application claim 14, Wu teaches contacting the RNA with a target double stranded DNA that is in vitro aka outside a cell, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1; PDF, Pg. 10, Col. 1, Labeling DNA oligos with N3-kethoaxl in vitro, meeting the DNA that is outside a cell in instant application claim 14.
Regarding instant application claim 15, Wu teaches contacting the RNA with a target double stranded DNA that is inside cells, see above and Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1; PDF, Pg. 10, Col. 1, KAS-seq – Col. 2, Low-input KAS-seq; PDF Pgs. 28-32, meeting the DNA that is inside a test cell in instant application claim 15.
Regarding instant application claim 16, Wu teaches the genome DNA, gDNA, bonded to the N3-kethoxal selective group is “subjected to biotinylation through ‘click’ chemistry before being fragmented”, i.e. click chemistry is cycloaddition, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1; PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting the cycloaddition in instant application claim 16.
Regarding instant application claim 17, Wu teaches “[t]he expression level of each gene was quantified with normalized fragments per kilobase of exon model per million reads mapped (FPKM) value with FPKM_count.pl in the RSeQC49 software”, see PDF, Pg. 10, Col. 2, RNA-seq data processing – Pg, 11, Col. 1, Ln. 10, meeting the quantifying the DNA fragment that contains the binding site in instant application claim 17.
Regarding instant application claims 19 and 20, Wu teaches a method of isolating DNA fragments by step 1.) isolating a single strand of a target DNA and contacting a target double stranded RNA polymerase II, such as Pol II, with N3-kethoxal aka kethoxal modified by the chemo selective group N3 in order to obtain/label single-stranded guanines at the site the Pol II binds in the genome, see Fig. 1 and below,
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, see also, Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting:
Step (h) in instant application claim 19; and,
The specific azido of N3 and the chemo selective group N3-kethoxal in instant application claim 20;
In step 2.) the chemo selective group is linked to a biotin binding member, see above and Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, where the genome DNA, gDNA, bonded to the N3-kethoxal selective group is “subjected to biotinylation through ‘click’ chemistry before being fragmented”, i.e. click chemistry is cycloaddition, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1, meeting step (i) in instant application claim 19;
In step 3.) the DNA is fragmented, see above and Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting step (j) in instant application claim 19;
The DNA single-stranded fragments are then enriched through the biotin–streptavidin interaction and subjected to library construction, followed by PCR -amplification and HT-sequencing, see above and Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting:
The method of isolating DNA fragments and step (k) in instant application claim 19.
Wu does not teach:
The instant application claim 1 limitations of a binding site for a guide-RNA of an RNA-guided endonuclease and step (a);
The instant application claim 19 limitation of step (g);
The guide-RNA or RNA-guided endonuclease in instant application claims 11, 14, 15, and 17; and,
The limitations of instant application claims 9, 10, and 13.
Zhang is in the known prior art field of methods and systems for amplifying and/or detecting target double-stranded or single-stranded nucleic acids through use of an amplification CRISPR system, see Abstract.
Regarding instant application claims 1 and 19, Zhang teaches a method comprising the steps of: (a) combining a sample comprising the target double-stranded nucleic acid with an amplification reaction mixture of (i) an amplification CRISPR system, (ii) a helicase, (iii) a primer pair comprising a first and second primer, and (iv) a polymerase; (b) amplifying the target nucleic acid; and (c) further amplifying the target nucleic acid by repeated opening, unwinding, annealing and extension under isothermal conditions; and, a step of detecting the target nucleic acid, see Paras. [0007]-[0008]. The amplification CRISPR system may comprise a first and second CRISPR/Cas complex, where the first CRISPR/Cas complex may comprise a first CRISPR/Cas enzyme and a first guide molecule that guides the first CRISPR/Cas complex to a first strand of the target nucleic acid and the second CRISPR/Cas complex may comprise a second CRISPR/Cas enzyme and a second guide molecule that guides the second CRISPR/Cas complex to a second strand of the target nucleic acid, see Paras. [0007]-[0008].
The CRISPR system amplification reaction mixture comprising a Cas9 protein contacted with a target double stranded genome DNA to cleave a single strand from the DNA at a target sight on the single stranded DNA, where the Cas9 protein may be modified with a guide-RNA, may not be modified at all aka unmodified, or may contain a differing modification in order to bond to the desired targeted site of the single stranded DNA, see Paras. [0023]-[0026];[0083]-[0085];[00107]-[00112];[00120]-[00127];[00142]-[00144]. The method combines a sample cell comprising the target double-stranded nucleic acid with the amplification reaction mixture that enters the sample cell in order to bind to the target site of the single stranded DNA, where the samples may be biological or environmental tissues, cells, and their progeny, see Paras. [0067]-[0068]; [00109]-[00112];[00167]-[00168];[00214]-[00218], meeting:
The binding site for a guide-RNA, the RNA-guided endonuclease, the guide-RNA and step (a) in instant application claim 1; and,
The binding site for a guide-RNA, the RNA-guided endonuclease without guide-RNA, a control cell, and step (g) in instant application claim 19.
Regarding instant application claims 11, 14, 15, and 17, Zhang teaches ““Nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses endonucleolytic catalytic activity for DNA cleavage”, see Para. [0071]. A “guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus”, where “proteins are RNA guided nucleases”, see Paras. [0081];[0094];[00142]-[00149], meeting the guide-RNA or the RNA-guided endonuclease in instant application claim 11, in instant application claim 14, in instant application claim 15, and in instant application claim 17.
Regarding instant application claims 9 and 10, Zhang teaches CRISPR/Cas enzyme may be active or dead, where the first and second CRISPR/Cas enzyme are catalytically active enzymes, such as the a Cas9, Casl2, Casl2b or Casl2c enzyme, and the dead CRISPR/Cas enzyme may be a dead Cas9 enzyme, a dead Casl2a, Casl2b, or Casl2c enzyme, see Paras. [0016];[0034]-[0035], modified with RNA guided nucleases, see Paras. [0081];[0094];[00142]-[00149], meeting the dead RNA guided nuclease and active RNA guided nuclease in instant application claim 9 and in instant application claim 10.
Regarding instant application claim 13, Zhang teaches “an engineered Cas9 protein as defined herein, such as Cas9, wherein the protein complexes with a nucleic acid molecule comprising RNA to form a CRISPR complex, wherein when in the CRISPR complex, the nucleic acid molecule targets one or more target polynucleotide loci, the protein comprises at least one modification compared to unmodified Cas protein, and wherein the CRISPR complex comprising the modified protein has altered activity as compared to the complex comprising the unmodified Cas9 protein”, see Paras. [00106]-[00108], where “cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions”, see Para. [00142], the guide RNA binding has “increased specificity for target polynucleotide loci as compared to off-target polynucleotide loci”, see Paras. [00106]; [00142], and “the CRISPR-Cas system [is] able to distinguish between target and off-target sequences that have greater than 80% to about 95% complementarity”, see Paras. [00142];[00149], meeting the off-target binding site identification for the RNA-guided endonuclease in instant application claim 13.
Regarding instant application claim 27, Wu teaches the use of a variety of kits associated with the kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach comprising isolating a single strand of a target DNA and contacting a target double stranded RNA polymerase II, such as Pol II, with N3-kethoxal aka kethoxal modified by the chemo selective group N3 in order to obtain/label single-stranded guanines at the site the Pol II binds in the genome, then the DNA single-stranded fragments are enriched through the biotin–streptavidin interaction and subjected to library construction, followed by PCR -amplification and HT-sequencing, see Abstract; Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting 2) and 3) in instant application claim 27.
Wu does not teach the instant application claim 27 limitations of a kit comprising 1) an RNA-guided endonuclease, 2) and 3).
Regarding instant application claim 27, Zhang teaches kits containing an RNA-guided endonuclease, guide-RNA, supports, primers, binding members such as “the immobilized reagent may be streptavidin and the labeled binding partner may be labeled biotin”, selective groups, such as “amine, azide”, and instructions for use of the kits, see Paras. [00142];[00150];[00203];[00262];[00301]-[00305], meeting the kit including 1) an RNA-guided endonuclease, 2), and 3) in instant application claim 27.
In reference to the above claims, it would have been obvious to one of ordinary
skill in the art, before the effective filing date of the claimed invention, to have modified the KAS-seq of Wu to use the KAS-seq in a CRISPR protein binding to single stranded DNA as taught by Zhang with a reasonable predictability of success for the purpose of efficiently identifying single stranded DNA generated by CRISPR protein binding to DNA in order to map regions of single stranded DNA generated by Cas9 binding, see Zhang, Paras. [0004]-[0005];[00106]-[00112];[00142];[00149].
The rationale to support a conclusion that the claim would have been obvious is that “a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely that product [was] not of innovation but of ordinary skill and common sense”, see MPEP 2143 I.E. Since patents are part of the literature of the prior art relevant for all they contain, see MPEP 2123, and both Wu and Zhang teach methods and systems for amplifying and/or detecting target single-stranded nucleic acids in the DNA sequencing industry, a person of ordinary skill in the art has good reason to modify Wu by relying upon Zhang before the effective filing date of the claimed invention for knowledge generally available within the DNA sequencing art regarding identifying single stranded DNA, see MPEP 2143 B & G and 2141, for the benefit of efficiently identifying single stranded DNA generated by CRISPR protein binding to DNA in order to map regions of single stranded DNA generated by Cas9 binding, see Zhang, Paras. [0004]-[0005];[00106]-[00112];[00142];[00149] and MPEP 2141.
As stated in Sakraida v. Ag Pro, Inc., 425 U.S. 273, 189 USPQ 449, reh’g denied,
426 U.S. 955 (1976), “[w]hen a work is available in one field of endeavor, design
incentives and other market forces can prompt variations of it, either in the same field
or a different one. If a person of ordinary skill can implement a predictable variation, §
103 likely bars its patentability. For the same reason, if a technique has been used to
improve one device, and a person of ordinary skill in the art would recognize that it
would improve similar devices in the same way, using the technique is obvious unless its
actual application is beyond his or her skill”, see MPEP 2141.
Selection of a known material, such as Cas9 applied in CRISPR instead of Pol II applied in ChIP-seq, based on its suitability for its intended use supported a prima facie obviousness determination in Sinclair & Carroll Co. v. Interchemical Corp., 325 U.S. 327, 65 USPQ 297 (1945), see MPEP 2144.07.
In addition, “[i]t is a settled principle of law that a mere carrying forward of an original patented conception involving only change of form, proportions, or degree, or the substitution of equivalents doing the same thing as the original invention, by substantially the same means,” such as Cas9 applied in CRISPR instead of Pol II applied in ChIP-seq, “is not such an invention as will sustain a patent, even though the changes of the kind may produce better results than prior inventions. In re Williams, 36 F.2d 436, 438, 4 USPQ 237 (CCPA 1929)”, see MPEP 2144.05.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 1-17, 19, 20, and 27 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 12428666 to He et al. (hereinafter He ‘666) in view of Zhang et al. (WO2020006036, published 02 January 2020, hereinafter Zhang) and Wu et al., (“Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ”, Published online 06 April 2020, Nature Methods, Vol. 17, Pgs. 515-523 and Supplementary PDF Pgs. 10-33, hereinafter Wu).
Regarding instant application claims 1 and 19, the claims of He ‘666 recite a method for isolating DNA fragments that contain a binding site for an RNA endonuclease, see claims 1 and 4-13, the method comprising: (a) contacting the RNA endonuclease with a target double-stranded DNA, thereby producing single stranded DNA (ss-DNA) at the site to which the RNA binds, see claims 5-10 and 13, (b) reacting the product of step (a) with kethoxal or an analog thereof, modified by a chemoselective group, thereby adding the chemoselective group to any unpaired guanine base within the ssDNA, see claims 1, 4, 5-10, and 13, (c) linking a first binding member of a specific binding pair to the chemoselective group added in step (b), see claims 10-12, (d) fragmenting the DNA after step (c) to produce fragmented target DNA, see claim 10, and (e) enriching for fragments that contain the first binding member of the specific binding pair by binding the product of step (d) to a support that comprises a second binding member of the specific binding pair, which specifically binds to the first binding member of the specific binding pair, see claims 10-12, meeting the method and most of steps (a)-(e) in instant application claim 1 and steps (g)-(j) in instant application claim 19.
Regarding instant application claims 2, 3, and 20, the claims of He ‘666 recite:
N3-kethoxal and the azido, see claims 1-4, 6, and 10, meeting in instant application claim 2 and in instant application claim 20; and,
The biotin-streptavidin pair, see claims 10-12, meeting instant application claim 3.
Regarding instant application claim 15, the claims of He ‘666 recite contacting the RNA endonuclease with a target double-stranded DNA that is inside a test cell, see claim 10, meeting most of instant application claim 15.
Regarding instant application claims 16 and 19, the claims of He ‘666 recite wherein linking the first binding member of the specific binding pair to the chemoselective group is performed via a cycloaddition reaction aka click chemistry, see claims 10-12, meeting instant application claim 16 and the cycloaddition in instant application claim 19.
The claims of He ‘666 do not recite:
The instant application claim 1 limitations of a binding site for a guide-RNA of an RNA-guided endonuclease, and step (a);
The instant application claim 19 step (g);
The guide-RNA or RNA-guided endonuclease in instant application claims 11, 14, 15, and 17; and,
The limitations of instant application claims 4-14, 17, and 27.
Regarding instant application claims 1 and 19, Zhang teaches a method comprising the steps of: (a) combining a sample comprising the target double-stranded nucleic acid with an amplification reaction mixture of (i) an amplification CRISPR system, (ii) a helicase, (iii) a primer pair comprising a first and second primer, and (iv) a polymerase; (b) amplifying the target nucleic acid; and (c) further amplifying the target nucleic acid by repeated opening, unwinding, annealing and extension under isothermal conditions; and, a step of detecting the target nucleic acid, see Paras. [0007]-[0008]. The amplification CRISPR system may comprise a first and second CRISPR/Cas complex, where the first CRISPR/Cas complex may comprise a first CRISPR/Cas enzyme and a first guide molecule that guides the first CRISPR/Cas complex to a first strand of the target nucleic acid and the second CRISPR/Cas complex may comprise a second CRISPR/Cas enzyme and a second guide molecule that guides the second CRISPR/Cas complex to a second strand of the target nucleic acid, see Paras. [0007]-[0008].
The CRISPR system amplification reaction mixture comprising a Cas9 protein contacted with a target double stranded genome DNA to cleave a single strand from the DNA at a target sight on the single stranded DNA, where the Cas9 protein may be modified with a guide-RNA, may not be modified at all aka unmodified, or may contain a differing modification in order to bond to the desired targeted site of the single stranded DNA, see Paras. [0023]-[0026];[0083]-[0085];[00107]-[00112];[00120]-[00127];[00142]-[00144]. The method combines a sample cell comprising the target double-stranded nucleic acid with the amplification reaction mixture that enters the sample cell in order to bind to the target site of the single stranded DNA, where the samples may be biological or environmental tissues, cells, and their progeny, see Paras. [0067]-[0068]; [00109]-[00112];[00167]-[00168];[00214]-[00218], meeting:
The binding site for a guide-RNA, the RNA-guided endonuclease, the guide-RNA and step (a) in instant application claim 1; and,
The binding site for a guide-RNA, the RNA-guided endonuclease without guide-RNA, a control cell, and step (g) in instant application claim 19.
Regarding instant application claims 11, 14, 15, and 17, Zhang teaches ““Nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses endonucleolytic catalytic activity for DNA cleavage”, see Para. [0071]. A “guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus”, where “proteins are RNA guided nucleases”, see Paras. [0081];[0094];[00142]-[00149], meeting the guide-RNA or the RNA-guided endonuclease in instant application claim 11, in instant application claim 14, in instant application claim 15, and in instant application claim 17.
Regarding instant application claims 9 and 10, Zhang teaches CRISPR/Cas enzyme may be active or dead, where the first and second CRISPR/Cas enzyme are catalytically active enzymes, such as the a Cas9, Casl2, Casl2b or Casl2c enzyme, and the dead CRISPR/Cas enzyme may be a dead Cas9 enzyme, a dead Casl2a, Casl2b, or Casl2c enzyme, see Paras. [0016];[0034]-[0035], modified with RNA guided nucleases, see Paras. [0081];[0094];[00142]-[00149], meeting the dead RNA guided nuclease and active RNA guided nuclease in instant application claim 9 and in instant application claim 10.
Regarding instant application claim 13, Zhang teaches “an engineered Cas9 protein as defined herein, such as Cas9, wherein the protein complexes with a nucleic acid molecule comprising RNA to form a CRISPR complex, wherein when in the CRISPR complex, the nucleic acid molecule targets one or more target polynucleotide loci, the protein comprises at least one modification compared to unmodified Cas protein, and wherein the CRISPR complex comprising the modified protein has altered activity as compared to the complex comprising the unmodified Cas9 protein”, see Paras. [00106]-[00108], where “cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions”, see Para. [00142], the guide RNA binding has “increased specificity for target polynucleotide loci as compared to off-target polynucleotide loci”, see Paras. [00106]; [00142], and “the CRISPR-Cas system [is] able to distinguish between target and off-target sequences that have greater than 80% to about 95% complementarity”, see Paras. [00142];[00149], meeting the off-target binding site identification for the RNA-guided endonuclease in instant application claim 13.
Regarding instant application claim 27, Zhang teaches kits containing an RNA-guided endonuclease, guide-RNA, supports, primers, binding members such as “the immobilized reagent may be streptavidin and the labeled binding partner may be labeled biotin”, selective groups, such as “amine, azide”, and instructions for use of the kits, see Paras. [00142];[00150];[00203];[00262];[00301]-[00305], meeting the kit including 1) an RNA-guided endonuclease, 2), and 3) in instant application claim 27.
Regarding instant application claim 27, Wu teaches the use of a variety of kits associated with the kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach comprising isolating a single strand of a target DNA and contacting a target double stranded RNA polymerase II, such as Pol II, with N3-kethoxal aka kethoxal modified by the chemo selective group N3 in order to obtain/label single-stranded guanines at the site the Pol II binds in the genome, then the DNA single-stranded fragments are enriched through the biotin–streptavidin interaction and subjected to library construction, followed by PCR -amplification and HT-sequencing, see Abstract; Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting 2) and 3) in instant application claim 27.
Regarding instant application claim 4, Wu teaches a method of isolating DNA fragments by step 1.) isolating a single strand of a target DNA and contacting a target double stranded RNA polymerase II, such as Pol II, with N3-kethoxal, i.e., kethoxal modified by the chemo selective group N3, in order to obtain/label single-stranded guanines at the site the Pol II binds in the genome, see Pg. 516, Fig. 1 and below,
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, see also, Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32. The DNA single-stranded fragments are then enriched through the biotin–streptavidin interaction and subjected to library construction, followed by PCR -amplification and HT-sequencing, see above and Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting the amplification in instant application claim 4.
Regarding instant application claim 5, Wu teaches the binding pair of biotin–streptavidin beads are present in the amplification because the PCR input is DNA or the beads suspension, see PDF Pgs. 31-32, Low-input KAS-seq, meeting the amplifying of the DNA with the binding pair in instant application claim 5.
Regarding instant application claim 6, Wu teaches washing the biotin–streptavidin beads 5 times with 1×binding and wash buffer prior to the PCR amplification of the beads suspension, see PDF Pgs. 31-32, Low-input KAS-seq, meeting the enrichment of the DNA with the binding pair in instant application claim 6.
Regarding instant application claim 7, Wu teaches isolating the DNA cells, such as with a 5 minute wash away, after N3-kethoxal labeling and before biotinylation, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting the washing before the first binding pair in instant application claim 7.
Regarding instant application claim 8, Wu teaches the target DNA is a genomic DNA, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting the target DNA in instant application claim 8.
Regarding instant application claims 11 and 12, Wu teaches after amplification the DNA fragment with the specific binding site undergoes HT-sequencing “on Illumina Nextseq500 platform with single-end 80-bp mode, aiming to get 30 million reads per library” aka a next generation sequencing method, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting:
The sequencing and the sequencing device in instant application claim 11 and in instant application claim 12.
Regarding instant application claim 14, Wu teaches contacting the RNA with a target double stranded DNA that is in vitro aka outside a cell, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1; PDF, Pg. 10, Col. 1, Labeling DNA oligos with N3-kethoaxl in vitro, meeting the DNA that is outside a cell in instant application claim 14.
Regarding instant application claim 17, Wu teaches “[t]he expression level of each gene was quantified with normalized fragments per kilobase of exon model per million reads mapped (FPKM) value with FPKM_count.pl in the RSeQC49 software”, see PDF, Pg. 10, Col. 2, RNA-seq data processing – Pg, 11, Col. 1, Ln. 10, meeting the quantifying the DNA fragment that contains the binding site in instant application claim 17.
In reference to the above claims, it would have been obvious to one of ordinary
skill in the art, before the effective filing date of the claimed invention, to have modified the method of labeling single stranded DNA recited in the claims of He ‘666 with the KAS-seq approach of Wu and to use the KAS-seq in a CRISPR protein binding to single stranded DNA as taught by Zhang with a reasonable predictability of success for the purpose of efficiently identifying single stranded DNA generated by CRISPR protein binding to DNA in order to map regions of single stranded DNA generated by Cas9 binding, see Zhang, Paras. [0004]-[0005];[00106]-[00112];[00142];[00149]; Wu, Abstract; Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1.
The rationale to support a conclusion that the claim would have been obvious is that “a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely that product [was] not of innovation but of ordinary skill and common sense”, see MPEP 2143 I.E. Since patents are part of the literature of the prior art relevant for all they contain, see MPEP 2123, and He ‘666, Wu, and Zhang all teach methods and systems for amplifying and/or detecting target single-stranded nucleic acids in the DNA sequencing industry, a person of ordinary skill in the art has good reason to modify the claims of He ‘666 by relying upon Wu and Zhang before the effective filing date of the claimed invention for knowledge generally available within the DNA sequencing art regarding identifying single stranded DNA, see MPEP 2143 B & G and 2141, for the benefit of efficiently identifying single stranded DNA generated by CRISPR protein binding to DNA in order to map regions of single stranded DNA generated by Cas9 binding, see Zhang, Paras. [0004]-[0005];[00106]-[00112];[00142];[00149]; Wu, Abstract; Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1; and MPEP 2141.
As stated in Sakraida v. Ag Pro, Inc., 425 U.S. 273, 189 USPQ 449, reh’g denied,
426 U.S. 955 (1976), “[w]hen a work is available in one field of endeavor, design
incentives and other market forces can prompt variations of it, either in the same field
or a different one. If a person of ordinary skill can implement a predictable variation, §
103 likely bars its patentability. For the same reason, if a technique has been used to
improve one device, and a person of ordinary skill in the art would recognize that it
would improve similar devices in the same way, using the technique is obvious unless its
actual application is beyond his or her skill”, see MPEP 2141.
Selection of a known material, such as Cas9 applied in CRISPR instead of Pol II applied in ChIP-seq, based on its suitability for its intended use supported a prima facie obviousness determination in Sinclair & Carroll Co. v. Interchemical Corp., 325 U.S. 327, 65 USPQ 297 (1945), see MPEP 2144.07.
In addition, “[i]t is a settled principle of law that a mere carrying forward of an original patented conception involving only change of form, proportions, or degree, or the substitution of equivalents doing the same thing as the original invention, by substantially the same means,” such as Cas9 applied in CRISPR instead of Pol II applied in ChIP-seq, “is not such an invention as will sustain a patent, even though the changes of the kind may produce better results than prior inventions. In re Williams, 36 F.2d 436, 438, 4 USPQ 237 (CCPA 1929)”, see MPEP 2144.05.
Claims 1-17, 19, 20, and 27 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 5-15 and 18-20 of copending Application No. 19302667 to He et al. (hereinafter He ‘667) in view of Zhang et al. (WO2020006036, published 02 January 2020, hereinafter Zhang) and Wu et al., (“Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ”, Published online 06 April 2020, Nature Methods, Vol. 17, Pgs. 515-523 and Supplementary PDF Pgs. 10-33, hereinafter Wu).
This is a provisional nonstatutory double patenting rejection.
Regarding instant application claims 1 and 19, the claims of He ‘667 recite a method for isolating DNA fragments that contain a binding site for an RNA endonuclease, see claims 6-20, the method comprising: (a) contacting the RNA endonuclease with a target double-stranded DNA, thereby producing single stranded DNA (ss-DNA) at the site to which the RNA binds, see claim 12, (b) reacting the product of step (a) with kethoxal or an analog thereof, modified by a chemoselective group, thereby adding the chemoselective group to any unpaired guanine base within the ssDNA, see claims 1, 2, 5-15, 18-20, (c) linking a first binding member of a specific binding pair to the chemoselective group added in step (b), see claims 12-15, 18-20, (d) fragmenting the DNA after step (c) to produce fragmented target DNA, see claims 12 and 18, and (e) enriching for fragments that contain the first binding member of the specific binding pair by binding the product of step (d) to a support that comprises a second binding member of the specific binding pair, which specifically binds to the first binding member of the specific binding pair, see claims 12-20, meeting the method and most of steps (a)-(e) in instant application claim 1 and steps (g)-(j) in instant application claim 19.
Regarding instant application claims 2, 3, and 20, the claims of He ‘667 recite:
N3-kethoxal and the azido, see claims 1, 2, 5, 7, meeting in instant application claim 2 and in instant application claim 20; and,
The biotin-streptavidin pair, see claims 12-14, meeting instant application claim 3.
Regarding instant application claim 14, the claims of He ‘667 recite contacting the RNA endonuclease with a target double-stranded DNA that is outside a test cell, see claim 6, meeting most of instant application claim 14.
Regarding instant application claim 15, the claims of He ‘667 recite contacting the RNA endonuclease with a target double-stranded DNA that is inside a test cell, see claims 12 and 18, meeting most of instant application claim 14.
Regarding instant application claims 16 and 19, the claims of He ‘667 recite wherein linking the first binding member of the specific binding pair to the chemoselective group is performed via a cycloaddition reaction aka click chemistry, see claims 12-15, meeting instant application claim 16 and the cycloaddition in instant application claim 19.
The claims of He ‘667 do not recite:
The instant application claim 1 limitations of a binding site for a guide-RNA of an RNA-guided endonuclease, and step (a);
The instant application claim 19 step (g);
The guide-RNA or RNA-guided endonuclease in instant application claims 11, 14, 15, and 17; and,
The limitations of instant application claims 4-13, 17, and 27.
Regarding instant application claims 1 and 19, Zhang teaches a method comprising the steps of: (a) combining a sample comprising the target double-stranded nucleic acid with an amplification reaction mixture of (i) an amplification CRISPR system, (ii) a helicase, (iii) a primer pair comprising a first and second primer, and (iv) a polymerase; (b) amplifying the target nucleic acid; and (c) further amplifying the target nucleic acid by repeated opening, unwinding, annealing and extension under isothermal conditions; and, a step of detecting the target nucleic acid, see Paras. [0007]-[0008]. The amplification CRISPR system may comprise a first and second CRISPR/Cas complex, where the first CRISPR/Cas complex may comprise a first CRISPR/Cas enzyme and a first guide molecule that guides the first CRISPR/Cas complex to a first strand of the target nucleic acid and the second CRISPR/Cas complex may comprise a second CRISPR/Cas enzyme and a second guide molecule that guides the second CRISPR/Cas complex to a second strand of the target nucleic acid, see Paras. [0007]-[0008].
The CRISPR system amplification reaction mixture comprising a Cas9 protein contacted with a target double stranded genome DNA to cleave a single strand from the DNA at a target sight on the single stranded DNA, where the Cas9 protein may be modified with a guide-RNA, may not be modified at all aka unmodified, or may contain a differing modification in order to bond to the desired targeted site of the single stranded DNA, see Paras. [0023]-[0026];[0083]-[0085];[00107]-[00112];[00120]-[00127];[00142]-[00144]. The method combines a sample cell comprising the target double-stranded nucleic acid with the amplification reaction mixture that enters the sample cell in order to bind to the target site of the single stranded DNA, where the samples may be biological or environmental tissues, cells, and their progeny, see Paras. [0067]-[0068]; [00109]-[00112];[00167]-[00168];[00214]-[00218], meeting:
The binding site for a guide-RNA, the RNA-guided endonuclease, the guide-RNA and step (a) in instant application claim 1; and,
The binding site for a guide-RNA, the RNA-guided endonuclease without guide-RNA, a control cell, and step (g) in instant application claim 19.
Regarding instant application claims 11, 14, 15, and 17, Zhang teaches ““Nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses endonucleolytic catalytic activity for DNA cleavage”, see Para. [0071]. A “guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus”, where “proteins are RNA guided nucleases”, see Paras. [0081];[0094];[00142]-[00149], meeting the guide-RNA or the RNA-guided endonuclease in instant application claim 11, in instant application claim 14, in instant application claim 15, and in instant application claim 17.
Regarding instant application claims 9 and 10, Zhang teaches CRISPR/Cas enzyme may be active or dead, where the first and second CRISPR/Cas enzyme are catalytically active enzymes, such as the a Cas9, Casl2, Casl2b or Casl2c enzyme, and the dead CRISPR/Cas enzyme may be a dead Cas9 enzyme, a dead Casl2a, Casl2b, or Casl2c enzyme, see Paras. [0016];[0034]-[0035], modified with RNA guided nucleases, see Paras. [0081];[0094];[00142]-[00149], meeting the dead RNA guided nuclease and active RNA guided nuclease in instant application claim 9 and in instant application claim 10.
Regarding instant application claim 13, Zhang teaches “an engineered Cas9 protein as defined herein, such as Cas9, wherein the protein complexes with a nucleic acid molecule comprising RNA to form a CRISPR complex, wherein when in the CRISPR complex, the nucleic acid molecule targets one or more target polynucleotide loci, the protein comprises at least one modification compared to unmodified Cas protein, and wherein the CRISPR complex comprising the modified protein has altered activity as compared to the complex comprising the unmodified Cas9 protein”, see Paras. [00106]-[00108], where “cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions”, see Para. [00142], the guide RNA binding has “increased specificity for target polynucleotide loci as compared to off-target polynucleotide loci”, see Paras. [00106]; [00142], and “the CRISPR-Cas system [is] able to distinguish between target and off-target sequences that have greater than 80% to about 95% complementarity”, see Paras. [00142];[00149], meeting the off-target binding site identification for the RNA-guided endonuclease in instant application claim 13.
Regarding instant application claim 27, Zhang teaches kits containing an RNA-guided endonuclease, guide-RNA, supports, primers, binding members such as “the immobilized reagent may be streptavidin and the labeled binding partner may be labeled biotin”, selective groups, such as “amine, azide”, and instructions for use of the kits, see Paras. [00142];[00150];[00203];[00262];[00301]-[00305], meeting the kit including 1) an RNA-guided endonuclease, 2), and 3) in instant application claim 27.
Regarding instant application claim 27, Wu teaches the use of a variety of kits associated with the kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach comprising isolating a single strand of a target DNA and contacting a target double stranded RNA polymerase II, such as Pol II, with N3-kethoxal aka kethoxal modified by the chemo selective group N3 in order to obtain/label single-stranded guanines at the site the Pol II binds in the genome, then the DNA single-stranded fragments are enriched through the biotin–streptavidin interaction and subjected to library construction, followed by PCR -amplification and HT-sequencing, see Abstract; Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting 2) and 3) in instant application claim 27.
Regarding instant application claim 4, Wu teaches a method of isolating DNA fragments by step 1.) isolating a single strand of a target DNA and contacting a target double stranded RNA polymerase II, such as Pol II, with N3-kethoxal, i.e., kethoxal modified by the chemo selective group N3, in order to obtain/label single-stranded guanines at the site the Pol II binds in the genome, see Pg. 516, Fig. 1 and below,
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, see also, Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32. The DNA single-stranded fragments are then enriched through the biotin–streptavidin interaction and subjected to library construction, followed by PCR -amplification and HT-sequencing, see above and Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting the amplification in instant application claim 4.
Regarding instant application claim 5, Wu teaches the binding pair of biotin–streptavidin beads are present in the amplification because the PCR input is DNA or the beads suspension, see PDF Pgs. 31-32, Low-input KAS-seq, meeting the amplifying of the DNA with the binding pair in instant application claim 5.
Regarding instant application claim 6, Wu teaches washing the biotin–streptavidin beads 5 times with 1×binding and wash buffer prior to the PCR amplification of the beads suspension, see PDF Pgs. 31-32, Low-input KAS-seq, meeting the enrichment of the DNA with the binding pair in instant application claim 6.
Regarding instant application claim 7, Wu teaches isolating the DNA cells, such as with a 5 minute wash away, after N3-kethoxal labeling and before biotinylation, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting the washing before the first binding pair in instant application claim 7.
Regarding instant application claim 8, Wu teaches the target DNA is a genomic DNA, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting the target DNA in instant application claim 8.
Regarding instant application claims 11 and 12, Wu teaches after amplification the DNA fragment with the specific binding site undergoes HT-sequencing “on Illumina Nextseq500 platform with single-end 80-bp mode, aiming to get 30 million reads per library” aka a next generation sequencing method, see Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1;PDF Pg. 10, Col. 1, Kas-seq; PDF Pg. 13, Extended Data Fig. 1; PDF Pgs. 28-32, meeting:
The sequencing and the sequencing device in instant application claim 11 and in instant application claim 12.
Regarding instant application claim 17, Wu teaches “[t]he expression level of each gene was quantified with normalized fragments per kilobase of exon model per million reads mapped (FPKM) value with FPKM_count.pl in the RSeQC49 software”, see PDF, Pg. 10, Col. 2, RNA-seq data processing – Pg, 11, Col. 1, Ln. 10, meeting the quantifying the DNA fragment that contains the binding site in instant application claim 17.
In reference to the above claims, it would have been obvious to one of ordinary
skill in the art, before the effective filing date of the claimed invention, to have modified the method of labeling single stranded DNA recited in the claims of He ‘667 with the KAS-seq approach of Wu and to use the KAS-seq in a CRISPR protein binding to single stranded DNA as taught by Zhang with a reasonable predictability of success for the purpose of efficiently identifying single stranded DNA generated by CRISPR protein binding to DNA in order to map regions of single stranded DNA generated by Cas9 binding, see Zhang, Paras. [0004]-[0005];[00106]-[00112];[00142];[00149]; Wu, Abstract; Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1.
The rationale to support a conclusion that the claim would have been obvious is that “a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely that product [was] not of innovation but of ordinary skill and common sense”, see MPEP 2143 I.E. Since patents are part of the literature of the prior art relevant for all they contain, see MPEP 2123, and He ‘667, Wu, and Zhang all teach methods and systems for amplifying and/or detecting target single-stranded nucleic acids in the DNA sequencing industry, a person of ordinary skill in the art has good reason to modify the claims of He ‘667 by relying upon Wu and Zhang before the effective filing date of the claimed invention for knowledge generally available within the DNA sequencing art regarding identifying single stranded DNA, see MPEP 2143 B & G and 2141, for the benefit of efficiently identifying single stranded DNA generated by CRISPR protein binding to DNA in order to map regions of single stranded DNA generated by Cas9 binding, see Zhang, Paras. [0004]-[0005];[00106]-[00112];[00142];[00149]; Wu, Abstract; Pg. 515, Col. 2, Genome-wide profiling of single-stranded DNA using N3-kethoxal-based labeling-Pg. 516, Col. 1, Ln. 7 and Fig. 1; and MPEP 2141.
As stated in Sakraida v. Ag Pro, Inc., 425 U.S. 273, 189 USPQ 449, reh’g denied,
426 U.S. 955 (1976), “[w]hen a work is available in one field of endeavor, design
incentives and other market forces can prompt variations of it, either in the same field
or a different one. If a person of ordinary skill can implement a predictable variation, §
103 likely bars its patentability. For the same reason, if a technique has been used to
improve one device, and a person of ordinary skill in the art would recognize that it
would improve similar devices in the same way, using the technique is obvious unless its
actual application is beyond his or her skill”, see MPEP 2141.
Selection of a known material, such as Cas9 applied in CRISPR instead of Pol II applied in ChIP-seq, based on its suitability for its intended use supported a prima facie obviousness determination in Sinclair & Carroll Co. v. Interchemical Corp., 325 U.S. 327, 65 USPQ 297 (1945), see MPEP 2144.07.
In addition, “[i]t is a settled principle of law that a mere carrying forward of an original patented conception involving only change of form, proportions, or degree, or the substitution of equivalents doing the same thing as the original invention, by substantially the same means,” such as Cas9 applied in CRISPR instead of Pol II applied in ChIP-seq, “is not such an invention as will sustain a patent, even though the changes of the kind may produce better results than prior inventions. In re Williams, 36 F.2d 436, 438, 4 USPQ 237 (CCPA 1929)”, see MPEP 2144.05.
Conclusion
No claims are allowed.
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/YO/Examiner, Art Unit 1692
/FEREYDOUN G SAJJADI/Supervisory Patent Examiner, Art Unit 1699