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 .
Application Status
This action is written in response to applicant’s correspondence received 8 June 2026. Claims 19, 23, 32-34, 36-41, and 43-46 are currently pending. Accordingly, claims 19, 23, 32-34, 36-41, and 43-46 are examined herein. The restriction requirement mailed 21 July 2022 is still deemed proper. Applicant's elected Group V and SEQ ID NOs: 27, 123, and 220 without traverse in the reply filed 30 September 2022.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The 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.
Claim(s) 19, 32-34, 41, and 43 is/are rejected under 35 U.S.C. 103 as being unpatentable over Barrangou (PG Pub No. WO 2016/033298 A1, published 3 March 2016) in view of Briner (Molecular cell 56.2 (2014): 333-339) and Cameron (PG Pub No. WO 2016/033246 A1).
Regarding claim 19, Barrangou is drawn to an invention concerned with methods and compositions comprising novel CRISPR polypeptides and polynucleotides for site-specific cleavage and nicking of nucleic acids (Abstract). Barrangou teaches a method of transcriptional control of a target DNA comprising contacting the target DNA with a chimeric sgRNA molecule and a deactivated Cas9 polypeptide (i.e., a Cas9 polypeptide that is unable to cleave target DNA but can bind to an sgRNA) that can bind to the target DNA, thereby controlling transcription of the target DNA (pg. 3, line 30 to pg. 4, line 4, pg. 9, lines 6-10; see FIG. 6C). Barrangou teaches that the chimeric sgRNA molecule comprises (a) a crRNA comprising a 3' region and a 5' region, wherein the 3' region comprises at least 10 consecutive nucleotides of a CRISPR repeat and the 5' region comprises at least 20 consecutive nucleotides of a spacer sequence located immediately upstream of the repeat (pg. 29, lines 17-27). Barrangou teaches that the chimeric sgRNA molecule comprises (b) a tracrRNA comprising a 5' and 3' region, wherein at least a portion of the 5' region of the tracrRNA is complementary to the 3' region of the crRNA and the 3' region of said tracrRNA forms secondary structures (e.g., hairpin structures) (pg. 29, lines 17-27). Barrangou teaches that the chimeric sgRNA molecule comprises, from 5’ to 3’, (i) a stem comprising a duplex between the 5’ end of the tracrRNA and the repeat of the crRNA, (ii) a nexus hairpin, and (iii) at least one terminal hairpin (pg. 8, lines 20-21; see Fig. 1). Barrangou teaches the use of a nexus hairpin having 100% identity to nucleotides 67-92 of the claimed SEQ ID NO: 428 (pg. 11, lines 9-11; see L. rhamnosus Cas9 sgRNA in Fig. 19 and in previously attached sequence alignment).
Barrangou does not teach or suggest that the nexus comprises at least one mutation selected from an insertion, substitution, and/or deletion of about one to five base pairs located from nucleotide 67 to nucleotide 92 of the claimed SEQ ID NO: 428 such that the GC content of the nexus hairpin is increased when compared to a construct comprising the nexus hairpin without at least one mutation (Claim 19). Briner does not teach or suggest that the synthetic nucleic acid construct binds to the target DNA but prevents complete cleavage of the target DNA by a wild-type Cas9 exhibiting DNA cleavage activity (Claim 19).
Briner is drawn to a study concerned with identifying and modifying crRNA:tracrRNA duplexes that direct Cas9 endonuclease cleavage activity (Abstract). Briner teaches that RNA- guided Cas9 endonucleases specifically target and cleave DNA in a sequence-dependent manner and have been widely used for programmable genome editing (Abstract). Briner teaches the use of a Cas9 sgRNA that comprises secondary structures including a stem, nexus hairpin, and at least one terminal hairpin created via the hybridization of a crRNA’s 3’ CRISPR repeat and tracrRNA’s 5’ region (pg. 336; see Figure 2). Briner teaches that the Cas9’s SgRNA nexus hairpins are relatively tolerant to sequence variations, including nucleotide substitutions, insertions, and deletions (i.e., an insertion, substitution, and/or deletion of at least one nucleotide in the nexus) (pg. 334; see Table S1 and green sgRNA in Figure 2). Briner teaches that the Cas9’s nexus can have additional cytosines and guanines inserted (i.e., the GC content of the sgRNA’s nexus was increased when compared to an sgRNA’s nexus not comprising the insertions in the nexus) (Table S1, variant 17). Briner teaches that the nexus is one of the most critical features of Cas9 sgRNAs and teaches that mutating the nexus hairpins opens new avenues for the exploitation of novel Cas9 proteins, with the potential to harness the diversity of natural Cas9 orthologs, including short Cas9 variants for convenient packaging and delivery (pg. 337).
Regarding the newly amended claim limitations, it is noted that the limitations are directed towards a functional limitation of the synthetic nucleic acid wherein the synthetic nucleic acid is capable of preventing cleavage of the target nucleic acid by a wild-type Cas9 exhibiting DNA cleavage activity. The newly amended claim limitations do not require the administration of the claimed wild-type Cas9 nor a newly recited specific structure that the synthetic nucleic acid must additionally comprise in order to perform the claimed function. Thus, the newly amended claim limitations are interpreted as being drawn towards a functional limitation of the synthetic nucleic acid construct that Applicant has incorporated in order to demonstrate a surprising result when compared to the previously recited references.
Cameron is drawn towards an invention concerned with methods for decreasing the number of off-target nucleic acid double-stranded breaks by Cas9 CRISPR systems (Abstract). Cameron teaches that dCa9 molecules can be utilized, alongside their sgRNA, to displace or prevent the binding of complexes comprising an active Cas9 (i.e., a wild-type Cas9) and its own sgRNA ([00139]; see FIG. 5). Cameron teaches that an active Cas9 may not cleave a nucleic acid that the dCas9 is bound to because the dCas9 prevents the active Cas9’s binding and cleavage activity such that, in this way, off-target binding and cleavage may be prevented ([00139]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the artificial sgRNA of Barrangou via the inclusion of at least one insertion in the nexus hairpin such that its GC content increased because it would have merely amounted to a combination of prior art elements according to known methods to yield predictable results. Because Briner teaches that increasing the GC content of the nexus via the insertion of one or more nucleotides did not abolish the Cas9’s sgRNA binding affinity for the target DNA, one would have expected inserting either a G or C in the nexus hairpin of Barrangou to have maintained the binding affinity of the Cas9’s sgRNA disclosed in Barrangou. Because both references teach the use of Cas9 sgRNA molecules, one would have expected the modification to have predictably resulted in the same functional outcome wherein the sgRNA binding affinity is not altered. Further, a person of ordinary skill in the art would have been motivated to have done so in order to explore the function of novel Cas9 orthologs that can bind to the mutant nexus hairpin sequence.
Additionally, based on the disclosure of Cameron, one of ordinary skill in the art would have expected that the dCas9 bound to the sgRNA rendered obvious by Barrangou and Briner to have been able to prevent cleavage of the target DNA by a wild-type Cas9 having cleavage activity because Cameron teaches that it was known in the art that dCas9 displaces active Cas9 molecules at the site where the dCas9 is bound to the target nucleic acid.
Regarding claims 41 and 43, Barrangou teaches that the sgRNA crRNA can be derived from Lactobacillus sp. (pg. 4, lines 20-26).
Regarding claims 32-34, Barrangou does not teach or suggest that the insertion, substitution, and/or deletion in the synthetic nucleic acid construct comprises replacing a pair of complementary base pairs with a pair of non-complementary nucleotides (Claim 32), replacing a complementary base pair with a different complementary base pair (Claim 33), or replacing a pair of non-complementary nucleotides with a different pair of non-complementary nucleotides (Claim 34).
Briner teaches that a Cas9 sgRNA stem can be mutated such that a complementary base pair is replaced with a pair of non-complementary base pairs (Table S1, variant 3). Briner teaches that the S. pyogenes stem can be mutated such that a complementary base pair is replaced with a different complementary base pair (Table S1, variant 2). Briner teaches that the S. pyogenes stem can be mutated such that non-complementary nucleotides are replaced with different non-complementary nucleotides (Table S1, variant 7). Briner teaches that the mutations in the Cas9 sgRNA constructs can facilitate specific DNA targeting with a previously orthologous Cas9 (pg. 335). Briner teaches that Cas9 sgRNAs have stems that can comprise variable nucleotides that can be substituted for at least three different bases (pg. 336; see green sgRNA in Figure 2).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the artificial Cas9 sgRNA of Barrangou via the inclusion of at least one insertion, substitution, and/or deletion in both the stem and nexus hairpin that replaces a pair of complementary base pairs with a pair of non-complementary nucleotides, replaces a complementary base pair with a different complementary base pair, or replaces a pair of non-complementary nucleotides with a different pair of non- complementary nucleotides, as described by Briner, because it would have merely amounted to a combination of prior art elements according to known methods to yield predictable results. Because Briner teaches that the stem of the Cas9 sgRNA is tolerant to sequence variations, one would have expected that modifying the sequence of Barrangou as described in the disclosure of Briner to have resulted in the generation of a functional sgRNA that could bind to the dCas9 of Barrangou. Further, because both references teach the use of Cas9 sgRNA molecules, one would have expected the modification to have predictably resulted in the same functional outcome wherein the sgRNA binding affinity is not altered.
Claim(s) 45 is/are rejected under 35 U.S.C. 103 as being unpatentable over Barrangou (PG Pub No. WO 2016/033298 A1, published 3 March 2016) in view of Briner (Molecular cell 56.2 (2014): 333-339) and Cameron (PG Pub No. WO 2016/033246 A1) as applied to claims 19, 32-34, 41, and 43 above, and further in view of Zhang (PG Pub No. US 2014/0186919).
Regarding claim 45, Barrangou in view of Briner and Cameron renders obvious claims 19, 32-34, 41, and 43 as described above.
Barrangou in view of Briner and Cameron does not teach or suggest that the synthetic nucleic acid construct comprises a sequence having about 95% identity to the claimed SEQ ID NO: 404 (Claim 45).
Zhang is drawn to an invention concerned with compositions and methods related to components of a CRISPR complex (Abstract). Zhang teaches the use of a synthetic Cas9 guide RNA that comprises a hairpin having 94.8% sequence identity to (i.e., has about 95% identity to) the claimed SEQ ID NO: 404 ([0091]; see FIG. 8C and SEQ ID NO: 106 in previously attached sequence alignment).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute a hairpin rendered obvious by Barrangou in view of Briner and Cameron for a hairpin having about 95% sequence identity to the claimed SEQ ID NO: 404 because it would have merely amounted to a simple substitution of one known element for another to obtain predictable results. Because both Barrangou and Zhang teach the use of sgRNAs comprising hairpin sequences that could bind to a Cas9 and target DNA, alongside disclosures that the hairpin sequence can be modified, one would have expected that substituting the hairpin sequence of the sgRNA of Barrangou for the sequence having about 95% identity to the claimed SEQ ID NO: 404 to have predictably resulted in the ability for the dCas9 of Barrangou to bind to a target nucleic acid.
Response to Arguments
Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Applicant alleges that the combination of Barrangou and Briner provides no evidence as to whether a mutant sgRNA would still be able to bind to a target DNA and control transcription of the same (Remarks; pg. 2).
This argument is not found persuasive because it is noted that the claims are not limited to contacting the target DNA with only the claimed synthetic nucleic acid construct. Rather, the claim goes on to further specify that the synthetic nucleic acid construct is introduced to a target DNA alongside a Cas9 polypeptide. Thus, the claim broadly reads on the use of a mutant sgRNA and a Cas9 molecule, wherein the combination of the sgRNA and Cas9 molecule has the function of being able to prevent cleavage of the target DNA by a wild-type Cas9 polypeptide exhibiting DNA cleavage activity.
As discussed above and as applied to claim 19, based on the disclosure of Cameron, one of ordinary skill in the art would have expected that the dCas9 bound to the sgRNA rendered obvious by Barrangou and Briner to have been able to prevent cleavage of the target DNA by a wild-type Cas9 having cleavage activity because Cameron teaches that it was known in the art that dCas9 displaces active Cas9 molecules at the site where the dCas9 is bound to the target nucleic acid. Thus, the newly amended claim limitations are expected and obvious in view of the combination of Barrangou, Briner, and Cameron.
Allowable Subject Matter
Regarding claims 23, 36-40, 44, and 46, the prior art does not teach the use of a synthetic nucleic acid construct comprising a stem that has at least 95% identity to the claimed SEQ ID NOs: 411-412, 414-415, 418, 420-421, 423, or 426 (see Claims 23 and 45).
The closest prior art, Barrangou (PG Pub No. US 2016/0345578 A1, published 1 December 2016) is drawn towards an invention concerned with CRISPR nucleic acids to screen for essential and non-essential genes (Abstract). Barrangou teaches the use of a synthetic Cas9 guide RNA that comprises a stem having 65.4% identity to the claimed SEQ ID NO: 414 ([0037]; see FIG. 16 and SEQ ID NO: 35 in previously attached sequence alignment).
However, Barrangou does not teach or suggest the use of a synthetic nucleic acid construct comprising a stem that has at least 95% identity to the claimed SEQ ID NOs: 411-412, 414-415, 418, 420-421, 423, or 426 (see Claims 23 and 45).
Therefore, the closest prior art does not teach or suggest the use of a synthetic nucleic acid construct comprising a stem that has at least 95% identity to the claimed SEQ ID NOs: 411-412, 414-415, 418, 420-421, 423, or 426. Accordingly, claims 23, 36-40, 44, and 46 are novel and nonobvious in view of the closest prior art.
The instant specification has provided evidence for the use of mutant sgRNA molecules comprising the claimed stem sequences comprising the claimed SEQ ID NOs and bind to target nucleic acids of interest through CRISPR targeting (pg. 5-6; see Figs. 2-3).
Thus, Applicant has provided adequate written description support for the claimed synthetic nucleic acids comprising the claimed SEQ ID NOs.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KYLE T REGA whose telephone number is (571)272-2073. The examiner can normally be reached Mon-Fri, 9AM-5PM (EDT/EST).
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Neil Hammell can be reached at 571-270-5919. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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