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
Because no certified translation has been filed for the CN2017105737520 Application in US Application No. 16/631,157, the effective filing date of the instant application is 12 April 2018.
Drawings
The drawings are objected to for the following reasons:
37 CFR 1.84 (u)(1) states “View numbers must be preceded by the abbreviation "FIG."”
In the current case, the view numbers for Figures 1-24 are preceded by the word "Figure" instead of the abbreviation "FIG.".
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claim 5 objected to because of the following informalities:
Regarding claim 5, the claim recites the phrase “the target nucleic acid molecules to be detected in the reaction system of the target nucleic acid molecules to be detected are obtained by amplification” in lines 2-3 of the claim. The phrase is grammatically incorrect and comprises a redundant iteration of “the target nucleic acid molecules to be detected”. Examiner suggests amending the phrase to recite “the target nucleic acid molecules to be detected in the reaction system
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.
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 1-9, 11, 13-19, 16-17, and 19-21 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.
Regarding claim 1, the claim recites the term “the system containing the target nucleic acid molecules to be detected” in lines 1-2 of the claim. There is insufficient antecedent basis for this limitation in the claim. It is unclear what system is being referred to or the requirements of the system.
Regarding claims 2, 11, 14, and 20-21, the claim recite a Cas protein “having an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a”. However, the criteria for evaluating of a Cas protein has similar collateral single-stranded DNA cleavage when compared to Cas12a is unclear. For example, is the claimed Cas protein’s activity similar to the collateral single-stranded DNA cleavage of Cas12a if the Cas protein collaterally cleaves single-stranded RNA? Is the claimed Cas protein’s activity similar if the Cas protein collaterally cleaves double-stranded DNA? Is the claimed Cas protein’s activity similar if the active site of the protein is similar to a RuvC site of a Cas12a, or may the active site of the claimed Cas protein be a different active site than RuvC? Because the specification does not describe the criteria for determining if a Cas protein has a similar activity to Cas12a, a person of ordinary skill in the art would not be able to ascertain the criteria for determining if a Cas protein has similar collateral single-stranded DNA cleavage when compared to Cas12a, as claimed.
Regarding claims 3 and 9, the claims recite the phrase “the target DNA”. There is insufficient antecedent basis for this limitation in the claims. It is unclear if the target DNA is further limiting the claimed “nucleic acid probe” or if the claimed target DNA is a different nucleic acid molecule.
Regarding claims 8-9 and 20-21, the claims are directed towards a “use” claim. Therefore, the claims are interpreted as being intended method claims. Accordingly, the claims are unclear because no active method steps are set forth in the claims.
Regarding claims 2, 4, and 9, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claim 15, the claim recites that “the cleavage is a trans-cleavage of the collateral single-stranded DNA” in lines 5-6 of the claim. However, the claim from which claim 15 depends (i.e., claim 11) does not recite “collateral single-stranded DNA” nor if the “target DNA” of claim 11 is a different DNA molecule than the “collateral single-stranded DNA” of claim 15 or if the “target DNA” of claim 11 is itself single-stranded.
Regarding claim 16, the claim recites that “the nucleic acid amplification” in lines 1-2 of the claim. However, the claim from which claim 15 depends (i.e., claim 15) does not recite a method of amplification. Therefore, claim 16 is unclear because it is not clear at what point amplification is performed in the claimed method of claim 15 or if claim 16 is meant to introduce a new method step.
Regarding claims 17 and 19, a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c).
In the present instance, claims 17 and 19 recite the broad recitation “(in the range of from -20nt to +20nt)” and the claim also recites “preferably in the range of from -15nt to +15nt, more preferably in the range of from -10nt to +l0nt)”,” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Regarding claims 2-7, as the claims are ultimately dependent on claim 1 and do not rectify the 35 USC 112(b) rejection above, the claims are also rejected under 35 USC 112(b).
Regarding claims 13 and 15-19, as the claims are ultimately dependent on claim 11 and do not rectify the 35 USC 112(b) rejection above, the claims are also rejected under 35 USC 112(b).
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 8-9 and 20-21 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter.
The claim(s) does/do not fall within at least one of the four categories of patent eligible subject matter because the claims are directed towards a “use” claim (i.e., a method claim) but do not recite any active methods steps. MPEP 2106.03 teaches that “a process defines ‘actions’, i.e., an invention that is claimed as an act or step, or a series of acts or steps.” Accordingly, the claims do not fall within at least one of the four categories of patent eligible subject matter because are unclear because no active method steps are set forth in the claims.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-2, 5-6, 8, 10, and 20 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Severinov (PG Pub No. US 2017/0321198 A1, published 9 November 2017, filed 7 April 2017).
Regarding claims 1, 8, 10, and 20, for the purposes of examination the claimed “nucleic acid probes” are interpreted as encompassing either DNA or RNA molecules that can be detected following their cleavage by a Cas protein. Further, the claimed “Cas protein “having an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a” is interpreted as encompassing a Cas protein that can collaterally cleave single-stranded RNA.
Severinov is directed towards an invention concerned with systems, methods, and compositions for targeting nucleic acids (Abstract). Severinov teaches the use of a method for detecting target nucleic acid molecules via the use of a nuclease assay (i.e., a collection of reagents encompassing a kit) that comprises a C2c2 guide RNA, a C2c2 protein (i.e., a Cas protein), and ssRNA molecules that can be detected following their collateral cleavage by the C2c2 protein (i.e., nucleic acid probes) ([1064]; see FIGs. 124A-124B). Severinov teaches that nuclease assays were performed in a nuclease assay buffer ([1083]).
Regarding claim 2,. Severinov teaches that C2c2 is able to collaterally cleave single-stranded RNA molecules that do not have complementarity to the C2c2 guide RNA ([1064]).
Regarding claim 5, Severinov teaches that the cleaved ssRNA molecules can be detected via an amplification method selected from PCR ([0730], [1064]; see FIG. 124B).
Regarding claim 6, Severinov teaches that a person of ordinary skill in the art could envisage the use of the C2c2 protein for targeting pathogenic RNA molecules in order to immunize subjects against RNA-only pathogens (i.e., the method can detect pathogenic microorganisms) ([0299], [0359]).
Claim(s) 1-4, 8-11, 13, 15, 20, 22-24, 30, 32, and 34-35 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Chen ("CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity." Science 360.6387 (29 November 2017): 436-439).
Regarding claims 1, 8-11, 15, 22-24, 32, and 34-35, Chen is directed towards a study concerned with single-stranded DNAse activity of CRISPR-Cas12a (Abstract). Chen teaches the use of an LbCas12a protein that is able to collaterally cleave single-stranded DNA probes that comprise a fluorophore quencher that emits fluorescence when the single-stranded DNA probe is cleaved through trans-cleavage of the probes when the Cas12a is bound to a target DNA molecule through a guide RNA and the protein’s RuvC domain (pg. 4, 6; see Figs. 2, 4a, and S2). Chen teaches that the cleavage of the probes is able to be identified via the detection of fluorescence emitted by the single-stranded DNA probes (i.e., the cleavage of the probes can identify if the target nucleic acid molecule is present in the sample) (pg. 6; see Fig. S8).
Regarding claim 2, Chen teaches the use of an LbCas12a protein (pg. 4, 6; see Figs. 2, 4a, and S2).
Regarding claim 3, Chen teaches that the guide RNA directs Cas12a to a target DNA molecule (pg. 4, 6; see Figs. 2, 4a, and S2).
Regarding claim 4, Chen teaches that the nucleic acid probes are single-stranded DNA (pg. 6; see Fig. S8).
Regarding claims 13 and 30, Chen teaches that the nucleic acid probe is single-stranded DNA with a fluorescent detectable label (pg. 6; see Fig. S8).
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) 16, 19, and 25-26 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chen ("CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity." Science 360.6387 (29 November 2017): 436-439) as applied to claims 1-4, 8-11, 13, 15, 20, 22-24, 30, 32, and 34-35 above, and further in view of Severinov (PG Pub No. US 2017/0321198 A1, published 9 November 2017, filed 7 April 2017).
Regarding claims 16, 19, and 25-26, Chen anticipates claims 1-4, 8-11, 13, 15, 20, 22-24, 30, 32, and 34-35 as described above.
Chen does not teach or suggest that a nucleic acid amplification method selected from PCR amplification is performed on the target nucleic acid probes (Claims 16 and 25-26). Chen does not teach or suggest that when the PAM is present at upstream or downstream of the target site, primers without a PAM sequence can be used (Claim 19).
However, one of ordinary skill in the art would have considered the teachings of Severinov as both references are common fields of endeavor pertaining to the use of Cas proteins that have collateral cleavage activity.
Severinov is directed towards an invention concerned with systems, methods, and compositions for targeting nucleic acids (Abstract). Severinov teaches the use of a method for detecting target nucleic acid molecules via the use of a nuclease assay (i.e., a collection of reagents encompassing a kit) that comprises a C2c2 guide RNA, a C2c2 protein (i.e., a Cas protein), and ssRNA molecules that can be detected following their collateral cleavage by the C2c2 protein (i.e., nucleic acid probes) ([1064]; see FIGs. 124A-124B). Severinov teaches that nuclease assays were performed in a nuclease assay buffer ([1083]). Severinov teaches that the cleaved nucleic acid probes can be detected via an amplification method selected from PCR ([0730], [1064]; see FIG. 124B). Severinov teaches that the cleaved nucleic acid probes can be detected via an amplification method selected from PCR (i.e., primers not containing a PAM sequence may be utilized to amplify collateral cleavage products) ([0730], [1064]; see FIG. 124B). Chen teaches that Cas12a PAM sequences are within 20 nucleotides of the target sequence (pg. 14; see Fig. 3).
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 method anticipated by Chen such that the target nucleic acid probes are amplified with primers, as described by Severinov. a person of ordinary skill in the art would have been motivated to do so in order to amplify the nucleic acid probes such that they can be detected. A person of ordinary skill in the art would have had a reasonable expectation of success because both Chen and Severinov teach the use of CRISPR proteins that have collateral single-stranded nucleic acid cleavage activity that can cleave and be used to detect the cleavage of single-stranded nucleic acid probes of interest.
Claim(s) 7, 12, and 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chen ("CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity." Science 360.6387 (29 November 2017): 436-439) as applied to claims 1-4, 8-11, 13, 15, 20, 22-24, 30, 32, and 34-35 above, and further in view of Shmakov (Molecular cell 60.3 (2015): 385-397).
Regarding claims 7, 12, and 21, for the purposes of examination the claimed “Cas protein having an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a” is interpreted as a Cas protein that mediates DNA cleavage in a 5’-PAM-dependent fashion analogous to Cas12a.
Chen anticipates claims 1-4, 8-11, 13, 15, 20, 22-24, 30, 32, and 34-35 as described above.
Chen does not teach or suggest that the Cas protein having an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a comprises C2c1 (Claims 7, 12, and 21).
However, one of ordinary skill in the art would have considered the teachings of Shmakov as both references are common fields of endeavor pertaining to the use of CRISPR Cas proteins.
Shmakov is directed towards a study concerned with diverse Class 2 CRISPR-Cas systems (Abstract). Shmakov teaches that C2c1 was a known CRISPR-Cas effector protein that is a Dual-RNA guided DNA endonuclease (Abstract; pg. 389). Shmakov teaches that C2c1 mediates DNA interference in a 5′-PAM-dependent fashion analogous to Cpf1 (i.e., the C2c1 Cas protein has an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a) (Abstract). Shmakov teaches a method of performing an in vitro cleavage assay comprising adding a guide RNA, a C2c1 Cas protein, and a DNA target in human cell lysate (pg. 391; see Figure 4). Shmakov teaches that the successful cleavage of the target DNA was able to be detected (pg. 391; see Figure 4).
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 the LbCas12a protein anticipated by Chen for a C2c1 protein having similar DNA cleavage activity, as described by Shmakov. A person of ordinary skill in the art would have had a reasonable expectation of success because Chen teaches the use of a LbCas12a protein that can collaterally cleave single-stranded DNA while Shmakov teaches that C2c1 mediated DNA cleavage in a fashion analogous to Cpf1 (i.e., Cas12a).
Claim(s) 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chen ("CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity." Science 360.6387 (29 November 2017): 436-439) as applied to claims 1-4, 8-11, 13, 15, 20, 22-24, 30, 32, and 34-35 above, and further in view of Kennedy (PG Pub No. US 2016/0040189 A1).
Regarding claim 14, the applicable teachings of Chen are discussed above as applied to claims 1-4, 8-11, 13, 15, 20, 22-24, 30, 32, and 34-35 above.
Chen does not specifically teach or suggest that the Cas protein is located in a first container and the nucleic acid probe is located in a separate container (Claim 14).
However, one of ordinary skill in the art would have considered the teachings of Kennedy as both references are common fields of endeavor pertaining to the use of kits comprising Cas proteins and nucleic acid probes.
Kennedy is directed towards an invention concerned with reagents and methods for increasing specificity and efficiency of RNA-guided genome editing. Kennedy teaches the use of containers that can contain an RNA-guided nuclease and a detection probe so that the individual elements can be proved into a reaction mixture in pre-measured use amounts ([0091]-[0092]).
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 system anticipated by Chen such that the Cas protein and the nucleic acid probe were in a first and second container, as described by Kennedy. A person of ordinary skill in the art would have been motivated to do so in order to utilize pre-determined amounts of the reagents such that a reaction mixture would consistently contain the same amounts of reagents. A person of ordinary skill in the art would have had a reasonable expectation of success because both Chen and Kennedy teach the use of Cas proteins and nucleic acid probes.
Claim(s) 17-18 and 27-29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chen ("CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity." Science 360.6387 (29 November 2017): 436-439) in view of Severinov (PG Pub No. US 2017/0321198 A1, published 9 November 2017, filed 7 April 2017) in view of as applied to claims 16, 19, and 25-26 above, and further in view of Mandell (PG Pub No. WO 2016/077350 A1).
Regarding claims 17-18 and 27-29, the applicable teachings of Chen in view of Severinov are discussed above as applied to claims 16, 19, and 25-26 above.
Chen further teaches that Cas12a PAM sequences are within 20 nucleotides of the target sequence (pg. 14; see Fig. 3).
Chen in view of Severinov does not specifically teach or suggest that when the PAM sequence is absent, nucleic acid amplification is carried out using primers introduced with a PAM sequence (Claim 17). Chen in view of Severinov does not specifically teach or suggest that the primer has the structure as claimed in Claim 18 (see Claim 18). Chen in view of Severinov does not specifically teach or suggest that the nucleic acids have been amplified using primers comprising a PAM sequence that is within 20 nucleotides upstream or downstream of the target sequence (Claims 27-29).
However, one of ordinary skill in the art would have considered the teachings of Mandell as both references are common fields of endeavor pertaining to the amplification of nucleic acids targeted and cleaved by CRISPR nucleases.
Mandell is directed towards an invention concerned with amplifying a target nucleic acid including providing a system having a crRNA or a derivative thereof, and a Cas protein or a variant thereof (Abstract). Mandell teaches the use of modified primers comprising CRISPR PAM sequences that can be utilized to introduce the PAM sequence to a target nucleic acid of interest via the amplification of a DNA fragment of interest via the primers ([00123]-[00124]; see FIG. 1). Mandell teaches that the primer comprises a PAM sequence that is flanked on both sides by a 5’ region and a 3’ region that is complementary to the DNA fragment ([00124]; see FIG. 1B-C). Mandell teaches that the CRISPR protein is able to target the PAM sequence present within the primer such that a DNA break is introduced at the DNA fragment that did not previously comprise the PAM sequence ([00124]).
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 primers used in the PCR amplification rendered obvious by Chen in view of Severinov such that, when a PAM sequence was absent from a target nucleic acid of interest, a primer comprising a PAM sequence flanked by a 5’ region and 3’ region that is complementary to the target nucleic acid of interest was utilized in order to introduce the PAM into the target nucleic acid of interest, as described by Mandell. A person of ordinary skill in the art would have been motivated to do so in order to introduce PAM sequences into nucleic acid probes that did not previously comprise PAM sequences such that the Cas12a may be targeted to the nucleic acid probes. A person of ordinary skill in the art would have had a reasonable expectation of success because both Mandell and Chen in view of Severinov teach the use of primers that can be used to amplify target nucleic acids comprising target sequences for CRISPR proteins.
Claim(s) 31 and 33 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chen ("CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity." Science 360.6387 (29 November 2017): 436-439) as applied to claims 1-4, 8-11, 13, 15, 20, 22-24, 30, 32, and 34-35 above, and further in view of You ("Measuring thermodynamic details of DNA hybridization using fluorescence." Biopolymers 95.7 (2011): 472-486).
Regarding claims 17-18 and 27-29, the applicable teachings of Chen are discussed above as applied to claims 1-4, 8-11, 13, 15, 20, 22-24, 30, 32, and 34-35 above.
Chen does not specifically teach or suggest that the fluorescent label comprises a HEX at the 5’ end and a BHQ1 group at the 3’ end (Claim 31). Chen does not specifically teach or suggest that the step of detecting fluorescence comprising using a fluorescence spectrometer (Claim 33).
However, one of ordinary skill in the art would have considered the teachings of You as both references are common fields of endeavor pertaining to the use of fluorescent quenching groups.
You is directed towards a study concerned with quenching groups that can be placed onto DNA molecules (Abstract). You teaches that utilizing a fluorescent label comprises a HEX at the 5’ end and a BHQ1 group at the 3’ end of a DNA molecule in order to detect the presence of the molecule via the use of a fluorescence spectrophotometer (pg. 474, 484; see Figure 4).
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 the fluorescence and quenching groups, alongside the detection method of the groups, anticipated by Chen for a HEX fluorescence group at the 5’ end and a BHQ1 group at the 3’ end of the DNA molecule, alongside the detection of the groups via the usage of a fluorescence spectrometer, as described by You. A person of ordinary skill in the art would have had a reasonable expectation of success because both You and Chen teach the use of fluorescence and quenching groups that can be utilized to detect target DNA molecules.
Claim(s) 1-8 and 10-13 are is/are rejected under 35 U.S.C. 103 as being unpatentable over Shmakov (Molecular cell 60.3 (2015): 385-397) in view of Mock (Nature Protocols 11.3 (2016): 598-615).
Regarding claim 1, Shmakov is directed towards a study concerned with diverse Class 2 CRISPR-Cas systems (Abstract). Shmakov teaches that C2c1 was a known CRISPR-Cas effector protein that is a Dual-RNA guided DNA endonuclease (Abstract; pg. 389). Shmakov teaches a method of performing an in vitro cleavage assay comprising adding a guide RNA, a C2c1 Cas protein, and a DNA target in human cell lysate (pg. 391; see Figure 4). Shmakov teaches that the in vitro cleavage assay was performed in a cleavage buffer (pg. 395). Shmakov teaches that the successful cleavage of the target DNA was able to be detected (pg. 391; see Figure 4).
Shmakov does not teach or suggest the use of a nucleic acid probe (Claims 1 and 11). Shmakov does not teach or suggest that the nucleic acid probe is single-stranded (Claim 4).
However, one of ordinary skill in the art would have considered the teachings of Mock as both references are common fields of endeavor pertaining to the analysis of double-stranded DNA that was edited via the use of a nuclease.
Mock is directed towards a study concerned with the use of two differently labeled probes that are placed within one amplicon at a gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles (Abstract). Mock teaches that although the use of CRISPR-Cas9 nucleases is known, quantification of resulting gene knockout rates induced by the CRISPR protein still remains a bottleneck (Abstract). Mock teaches that the two probes are single-stranded nucleic acids that are complementary to a region of target dsDNA and that, following NHEJ induced by edits induced by designer nucleases, the successful detection of NHEJ affected sequences could be detected (pg. 600; see 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 substitute the method of detecting the cleavage of target DNA described in Shmakov for an alternative detection method of detecting the cleavage of target DNA that utilizes single-stranded nucleic acid probes, as described by Mock. A person of ordinary skill in the art would have been motivated to do so in order to detect NHEJ events that were mediated by the C2c1 nuclease of Shmakov. A person of ordinary skill in the art would have had a reasonable expectation of success because both Shmakov and Mock teach that CRISPR nucleases are known to edit dsDNA and Mock teaches that NHEJ events mediated by the editing of the dsDNA is able to be detected via the use of single-stranded nucleic acid probes.
Regarding claims 2 and 11, for the purposes of examination the claimed “Cas protein having an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a is interpreted as a Cas protein that mediates DNA cleavage in a 5’-PAM-dependent fashion analogous to Cas12a. Shmakov teaches that C2c1 mediates DNA interference in a 5′-PAM-dependent fashion analogous to Cpf1 (i.e., the C2c1 Cas protein has an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a) (Abstract).
Regarding claim 3, Shmakov teaches that the guide RNA refers to an RNA molecule that directs the C2c1 protein to bind to the target DNA (pg. 391; see Figure 4).
Regarding claim 5, Shmakov teaches that the target DNA was amplified via PCR prior to its utilization in the cleavage assay (pg. 389).
Regarding claim 6, Shmakov teaches that the C2c1 guide RNA is able to detect specific target DNA selected from a human EMX1 locus (pg. 391; see Figure 4).
Regarding claims 7 and 12, Shmakov teaches that the Cas protein comprises C2c1 (pg. 391; see Figure 4).
Regarding claim 8, the claim is interpreted as being directed towards a method of using the claimed Cas protein to detect target nucleic acid molecules. Shmakov teaches a method of performing an in vitro cleavage assay comprising adding a guide RNA, a C2c1 Cas protein, and a DNA target (i.e., a nucleic acid probe) in human cell lysate (pg. 391; see Figure 4). Shmakov teaches that the successful cleavage of the target DNA was able to be detected (pg. 391; see Figure 4).
Regarding claim 10, Shmakov teaches a method of performing an in vitro cleavage assay comprising adding a guide RNA, a C2c1 Cas protein, and a DNA target (i.e., a nucleic acid probe) in human cell lysate (i.e., adding a kit comprising a guide RNA, a Cas protein, and a nucleic acid probe to human cell lysate) (pg. 391; see Figure 4).
Regarding claim 13, Mock teaches that the two probes are single-stranded nucleic acids that are complementary to a region of target dsDNA and that, following NHEJ induced by edits induced by designer nucleases, the successful detection of NHEJ affected sequences could be detected via the use of fluorescent detectable labels (pg. 600; see Figure 2).
Claim(s) 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Shmakov (Molecular cell 60.3 (2015): 385-397) in view of Mock (Nature Protocols 11.3 (2016): 598-615) as applied to claims 1-8 and 10-13 above, and further in view of Kennedy (PG Pub No. US 2016/0040189 A1).
Regarding claim 14, Shmakov in view of Mock renders obvious claims 1-8 and 10-13 as described above.
Shmakov in view of Mock does not specifically teach or suggest that the Cas protein is located in a first container and the nucleic acid probe is located in a separate container (Claim 14).
However, one of ordinary skill in the art would have considered the teachings of Kennedy as both references are common fields of endeavor pertaining to the use of kits comprising Cas proteins and nucleic acid probes.
Kennedy is directed towards an invention concerned with reagents and methods for increasing specificity and efficiency of RNA-guided genome editing. Kennedy teaches the use of containers that can contain an RNA-guided nuclease and a detection probe so that the individual elements can be proved into a reaction mixture in pre-measured use amounts ([0091]-[0092]).
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 system rendered obvious by Shmakov in view of Mock such that the Cas protein and the nucleic acid probe were in a first and second container, as described by Kennedy. A person of ordinary skill in the art would have been motivated to do so in order to utilize pre-determined amounts of the reagents such that a reaction mixture would consistently contain the same amounts of reagents. A person of ordinary skill in the art would have had a reasonable expectation of success because both the Shmakov in view of Mock and Kennedy teach the use of Cas proteins and nucleic acid probes.
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 and 19-35 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-12, 14-15, and 25 of U.S. Patent No. 12,180,539.
Although the claims at issue are not identical, they are not patentably distinct from each other because the patented claims anticipate the instant claims.
Regarding claims 1, 20, 22-23, patented claim 1 recites a method for detecting target nucleic acid molecules comprising a target sequence, the method comprising steps of:
1) Adding (a) a guide RNA comprising a target site that specifically hybridizes with the target sequence; (b) a Cas12 protein having collateral cleavage activity, wherein the collateral cleavage activity of the Cas 12 protein is activated when specific hybridization of the guide RNA and the target sequence occurs; (c) a detectably labeled nucleic acid probe susceptible to the collateral cleavage activity of the Cas12 protein; and (d) a buffer into a system containing the target nucleic acid molecules to be detected, and
2) then detecting cleavage of the nucleic acid probe.
It is noted that the patented nucleic acid probe is a single-stranded DNA molecule as defined by the patented specification (Col. 3, lines 11-17).
Regarding claims 2 and 24, patented claim 11 recites identical claim limitations.
Regarding claim 3, patented claim 1 recites that the guide RNA directs the Cas protein to the target DNA.
Regarding claims 4 and 30-31, patented claims 9-10 recite that the nucleic acid probe comprise a fluorescent HEX group and a BHQ1 quenching group.
Regarding claims 5 and 25, patented claim 2 recites identical limitations.
Regarding claim 6, patented claim 12 recites that the target nucleic acid comprises a sequence found in a pathogen.
Regarding claims 7 and 21, patented claim 11 recites that the Cas12 protein is C2c1.
Regarding claims 8-9, patented claim 1 claims the use of a Cas12 protein having collateral cleavage activity that is activated when specific hybridization of the guide RNA and the target sequence occurs.
Regarding claims 10, 14, and 34-35, patented claim 25 claims a kit comprising i) a first container and a Cas12 protein located in the first container, wherein the Cas12 protein has collateral cleavage activity; ii) a second container and a guide RNA located in the second container, wherein the guide RNA that specifically hybridizes with the target sequence, wherein the collateral cleavage activity is activated when the specific hybridization occurs; iii) a third container and a detectably labeled nucleic acid probe located in the third container, wherein the detectably labeled nucleic acid probe is susceptible to the collateral cleavage activity of the Cas12 protein; and iv) a fourth container and a buffer located in the fourth container.
Regarding claim 11, patented claim 14 claims a detection system comprising: (a) target nucleic acid molecules comprising a target sequence of interest; (b) a Cas12 protein having collateral cleavage activity; (c) a guide RNA comprising a target site that specifically hybridizes with the target sequence of interest, wherein the collateral cleavage activity is activated when the specific hybridization occurs; (d) a detectably labeled nucleic acid probe susceptible to the collateral cleavage activity of the Cas12 protein; and (e) a buffer.
Regarding claim 12, patented claim 15 claims that the Cas protein is C2c1.
Regarding claim 13, patented claim 14 claims that the nucleic acid probe comprised a detectable label.
Regarding claim 15, patented claim 1 recites that the method can be utilized to detect (i.e., determine the presence or absence of) a target nucleic acid molecules comprising a target sequence.
Regarding claims 16 and 26, patented claim 3 recites identical limitations.
Regarding claim 17 and 27-28, patented claims 4-5 claim that the target nucleic acid molecules are amplified using primers comprising a protospacer adjacent motif (PAM) sequence that are upstream or downstream of a target site of the guide RNA. Patented claim 20 claims that the PAM site is +20 nt to -20 nt upstream or downstream of the target site of the guide RNA.
Regarding claims 19 and 29, patented claim 6 recites identical limitations.
Regarding claim 32, patented claim 7 recites identical limitations.
Regarding claim 33, patented claim 8 claims identical limitations.
Claim 18 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-12, 14-15, and 25 of U.S. Patent No. 12,180,539 as applied to claims 1-17 and 19-35 above, further in view of Stadhouders (The Journal of Molecular Diagnostics 12.1 (2010): 109-117).
Regarding claim 18, patented claims 1-12, 14-15, and 25 anticipate claims 1-17 and 19-35 as described above.
Patented claims 1-12, 14-15, and 25 do not specifically teach or suggest that the primer introduced with PAM has a structure of formula I from 5’ to 3’: P1-P2-P3 wherein, P1 is a 5' segment sequence complementary or non-complementary to the sequence of the target nucleic acid molecule at the 5' end; P2 is a PAM sequence; and P3 is a 3' segment sequence complementary to the sequence of the target nucleic acid molecule at the 3' end (Claim 18).
However, one of ordinary skill in the art would have considered the teachings of Stadhouders as both references are common fields of endeavor pertaining to the use of primers in nucleic acid amplification methods.
Stadhouders is drawn towards a study concerned with the effect of primer-template mismatches on amplification of a template nucleic acid (Abstract). Stadhouders teaches that mismatches located in the 3′ end region of the primer are known to be exceptionally detrimental to PCR priming (pg. 115).
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 patented primer such that the primer comprising the PAM sequence was complementary to the target nucleic acid at the 3’ end, as described by Stadhouders. A person of ordinary skill in the art would have been motivated to do so in order to ensure that successful PCR priming and subsequent amplification could take place. A person of ordinary skill in the art would have had a reasonable expectation of success because both the patented claims and Stadhouders teach the use of primers utilized for the amplification of a target nucleic acid.
Claims 1-13, 15-17, and 19-35 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 4-8, 10, and 12 of U.S. Patent No 11,584,955 B2.
Although the claims at issue are not identical, they are not patentably distinct from each other because the patented claims anticipate the instant claims.
Regarding claims 1, 20, and 22-23, patented claim 1 recites a method for detecting a target nucleic acid molecule that contains a target sequence of interest and is in a sample, the method comprising steps of:
(a) providing a mixture including:
a sample that might contain the target sequence of interest;
a Cas12 protein having collateral cleavage activity that is increased when the Cas12 protein is complexed with a guide RNA that is specifically hybridized with the target sequence of interest;
a guide RNA that specifically hybridizes with the target sequence of interest wherein, if the sample contains the target sequence of interest and the guide RNA hybridizes therewith, the collateral cleavage activity of the Cas12 protein is increased;
a nucleic acid probe susceptible to the collateral cleavage activity, so that the nucleic acid probe has a first uncleaved state and a second cleaved state, and it transitions from the first uncleaved state to the second cleaved state when cleaved by the Cas12 protein, the nucleic acid probe being detectably labeled so that its transition from the first state to the second state is detectable; and
a buffer;
It is noted that the patented nucleic acid probe is a single-stranded DNA molecule as defined by the patented specification (Col. 3, lines 11-17).
Regarding claims 2 and 24, patented claim 2 recites identical claim limitations.
Regarding claim 3, patented claim 1 recites that the guide RNA directs the Cas protein to the target DNA.
Regarding claims 4 and 30-31, patented claims 8 and 12 recite that the nucleic acid probe comprise a fluorescent HEX group and a BHQ1 quenching group.
Regarding claims 5 and 25, patented claim 1 recites identical limitations.
Regarding claim 6, patented claim 1 recites that the method can detect specific target DNA.
Regarding claims 7 and 21, patented claim 2 recites that the Cas12 protein is C2c1.
Regarding claims 8-9, patented claim 1 claims the use of a Cas12 protein having collateral cleavage activity that is activated when specific hybridization of the guide RNA and the target sequence occurs.
Regarding claims 10-11 and 34-35, patented claim 1 claims the use of a detection system comprising a combination of a guide RNA, a Cas protein, and a ssDNA nucleic acid probe.
Regarding claim 12, patented claim 2 claims that the Cas protein is C2c1.
Regarding claim 13, patented claim 8 claims that the nucleic acid probe comprised a fluorescent group (i.e., a detectable label).
Regarding claim 15, patented claim 1 recites that the method can be utilized to detect (i.e., determine the presence or absence of) a target nucleic acid molecules comprising a target sequence.
Regarding claims 16 and 26, patented claim 4 recites identical limitations.
Regarding claim 17 and 27-28, patented claims 5-7 claim that the target nucleic acid molecules are amplified using primers comprising a protospacer adjacent motif (PAM) sequence that are upstream or downstream of a target site of the guide RNA. Patented claim 20 claims that the PAM site is +20 nt to -20 nt upstream or downstream of the target site of the guide RNA.
Regarding claims 19 and 29, patented claim 7 recites identical limitations.
Regarding claim 32, patented claim 9 recites identical limitations.
Regarding claim 33, patented claim 10 claims identical limitations.
Claim 14 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 4-8, 10, and 12 of U.S. Patent No 11,584,955 B2 as applied to claims 1-13, 15-17, and 19-35 above, further in view of Kennedy (PG Pub No. US 2016/0040189 A1).
Regarding claim 14, patented claims 1-2, 4-8, 10, and 12 anticipate claims 1-17 and 19-35 as described above.
Patented claims 1-2, 4-8, 10, and 12 do not specifically teach or suggest that the Cas protein is located in a first container and the nucleic acid probe is located in a separate container (Claim 14).
However, one of ordinary skill in the art would have considered the teachings of Kennedy as both references are common fields of endeavor pertaining to the use of kits comprising Cas proteins and nucleic acid probes.
Kennedy is directed towards an invention concerned with reagents and methods for increasing specificity and efficiency of RNA-guided genome editing. Kennedy teaches the use of containers that can contain an RNA-guided nuclease and a detection probe so that the individual elements can be proved into a reaction mixture in pre-measured use amounts ([0091]-[0092]).
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 patented claims such that the Cas protein and the nucleic acid probe were in a first and second container, as described by Kennedy. A person of ordinary skill in the art would have been motivated to do so in order to utilize pre-determined amounts of the reagents such that a reaction mixture would consistently contain the same amounts of reagents. A person of ordinary skill in the art would have had a reasonable expectation of success because both the patented claims and Kennedy teach the use of Cas proteins and nucleic acid probes.
Claim 18 rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 4-8, 10, and 12 of U.S. Patent No 11,584,955 B2 as applied to claims 1-13, 15-17, and 19-35 above, further in view of Stadhouders (The Journal of Molecular Diagnostics 12.1 (2010): 109-117).
Regarding claim 18, patented claims 1-2, 4-8, 10, and 12 anticipate claims 1-17 and 19-35 as described above.
Patented claims 1-2, 4-8, 10, and 12 do not specifically teach or suggest that the primer introduced with PAM has a structure of formula I from 5’ to 3’: P1-P2-P3 wherein, P1 is a 5' segment sequence complementary or non-complementary to the sequence of the target nucleic acid molecule at the 5' end; P2 is a PAM sequence; and P3 is a 3' segment sequence complementary to the sequence of the target nucleic acid molecule at the 3' end (Claim 18).
However, one of ordinary skill in the art would have considered the teachings of Stadhouders as both references are common fields of endeavor pertaining to the use of primers in nucleic acid amplification methods.
Stadhouders is drawn towards a study concerned with the effect of primer-template mismatches on amplification of a template nucleic acid (Abstract). Stadhouders teaches that mismatches located in the 3′ end region of the primer are known to be exceptionally detrimental to PCR priming (pg. 115).
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 patented primer such that the primer comprising the PAM sequence was complementary to the target nucleic acid at the 3’ end, as described by Stadhouders. A person of ordinary skill in the art would have been motivated to do so in order to ensure that successful PCR priming and subsequent amplification could take place. A person of ordinary skill in the art would have had a reasonable expectation of success because both the patented claims and Stadhouders teach the use of primers utilized for the amplification of a target nucleic acid.
Subject Matter Eligibility
Regarding claims 9, 15, 20, 22, and 34, Applicant provides adequate 35 USC 112(a) support for the claimed Cas12 proteins comprising collateral cleavage activity (Instant specification; pg. 11, 13-14, 33-40; see Examines 14-15 and Figs. 8-9, 12 and 22-24). Applicant has demonstrated that nine species of Cas12a proteins had both cis and trans cleavage activity on ssDNA (pg. 39; see Figs. 4C and 4D). Applicant has identified key sites and mechanisms of the cis and trans cleavage of ssDNA by Cas12a (pg. 39-40). Applicant has demonstrated that Cas12b has both cis and trans cleavage activity and identified key mechanisms of Cas12b collateral cleavage activity (pg. 37-38).
Regarding dependent claims 16-19, 21, 23-33, and 35, as the claims are dependent on claims 15, 20, 22, and 34, the claims have adequate written description support for the same reasons discussed above as applied to claims 9, 15, 20, 22, and 34.
Conclusion
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/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636