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 .
Election/Restrictions
Applicant’s election without traverse of group I, claims 1-4, 6-10, 12-14, 16-21, 23-25, 27, and 49-50 in the reply filed on 12/4/2025 is acknowledged.
The response continues by providing arguments, “Hsu's paragraph [0013] describes generating multiple gRNAs from a single array, not the spatial segregation of distinct sets of guide molecules into individual discrete volumes as required by the pending claims.” This argument has been thoroughly reviewed but is not considered persuasive as this is not a limitation of the product claims. Thus this is not persuasive as it is inconsistent with the claims.
Claims 30-31, 33-35. 37-39, 41, 43, 46-47 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 12/4/2025
Priority
The instant application was filed 12/03/2021 and is a national stage entry of PCT/US2021/022845 with an international filing date: 03/17/2021 and claims priority from provisional application 62991004 , filed 03/17/2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 12/3/2021, 2/26/2024, 10/22/2024 are being considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Objections
Claims 1-4, 6-10, 12-14, 16-21, 23-25, 27, and 49-50 are objected to because of the following informalities:
Claim 1 recites, “a.”, “b.”, “c.”, “d.”, “e.” MPEP 608.01(m) states: Each claim begins with a capital letter and ends with a period. Periods may not be used elsewhere in the claims except for abbreviations. See Fressola v.Manbeck, 36 USPQ2d 1211 (D.D.C. 1995).
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-4, 6-10, 12-14, 16-21, 23-25, 27, and 49-50 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
As set forth in In re Alonso 88 USPQ2d 1849 (Fed. Cir. 2008), at 1851:
The written description requirement of 35 U.S.C. § 112, ¶ 1, is straightforward: “The specification shall contain a written description of the invention ….” To satisfy this requirement, the specification must describe the invention in sufficient detail so “that one skilled in the art can clearly conclude that the inventor invented the claimed invention as of the filing date sought.” Lockwood v. Am. Airlines, Inc., 107 F.3d 1565, 1572 [41 USPQ2d 1961] (Fed. Cir. 1997); see also LizardTech, Inc. v. Earth Res. Mapping, Inc., 424 F.3d 1336, 1345 [76 USPQ2d 1724] (Fed. Cir. 2005); Eiselstein v. Frank, 52 F.3d 1035, 1039 [34 USPQ2d 1467] (Fed. Cir. 1995).
Alonso at 1852:
A genus can be described by disclosing: (1) a representative number of species in that genus; or (2) its “relevant identifying characteristics,” such as “complete or partial structure, other physical and/or chemical properties, functional characteristics when coupled with a known or disclosed correlation between function and structure, or some combination of such characteristics.” Enzo, 323 F.3d at 964.
In applying the test as set forth in Alonso, it is noted that applicant is claiming A high throughput method for detecting target molecules comprising
a. generating sets of guide molecules, each set of guide molecules comprising guide molecules capable of binding one or more target sequences of a target molecule and designed to form a complex with a Cas protein;
b. distributing a plurality of sets of guide molecules, thereby spatially segregating each set of guide molecules;
c. distributing to each set of guide molecules a sample solution, detection reagents, and a reporter construct comprising a non-target sequence, and a Cas protein
d. initiating a detection reaction, wherein the Cas protein cleaves the non-target sequence of the reporter constructs once activated by the target sequences; and
e. measuring the signal generated from the reporter construct associated with each set of guide molecules from cleavage of the non-target sequence of the reporter constructs.
Thus the claims encompass anything which can be considered a guide molecule by any standard which can be considered the capable of binding a target sequence of a target molecule by any standard.
The claims encompass anything can be considered a target molecule by any standard.
Further the claim requires, “wherein the Cas protein cleaves the non-target sequence of the reporter constructs once activated by the target sequences.” The specification does not provide a specific definition or standard to differentiate a target sequence from a non-target sequence.
Dependent claims limit the guide molecule to RNA. Thus claim 1 encompasses additional guide molecules for the dependent claim to properly limit.
The teachings of the specification appear to be limited to the use of single stranded RNA as the guide molecules and RNA or DNA as target molecules.
Thus the teachings of the specification are limited to a single species of guide molecules and two species of target molecules and target sequences, which do not provide antecedent basis for the breadth of the claims. Thus the claims lack adequate written description.
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 1-4, 6-10, 12-14, 16-21, 23-25, 27, and 49-50 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.
Claim 1 is indefinite because it lacks a positive active step relating back to the preamble. The preamble recites a method high throughput method for detecting target molecules, however the last positive active step is drawn to measuring the signal generated from the reporter construct associated with each set of guide molecules from cleavage of the non-target sequence of the reporter constructs. Therefore it is unclear as to whether the method is drawn to high throughput method for detecting target molecules or measuring the signal generated from the reporter construct associated with each set of guide molecules from cleavage of the non-target sequence of the reporter constructs.
Claim 1 requires, “each set of guide molecules comprising guide molecules capable of binding one or more target sequences of a target molecule and designed to form a complex with a Cas protein” The claim later requires, “ initiating a detection reaction, wherein the Cas protein cleaves the non-target sequence of the reporter constructs once activated by the target sequences.” CRISPR/Cas proteins can detect single nucleotide differences in RNA or DNA sequences. Thus the claim is confusing and unclear how a non-target sequence is cleaved.
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.
Claim(s) claims 1-4, 6-10, 12, 14, 16-21, 23-25, 27, and 49-50is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Collins (WO2018170340).
With regards to claim 1, Collins teaches a high throughput method for detecting target molecules (para [0026] "the invention provides a method for detecting target RNAs in samples") comprising a. generating sets of guide molecules, each set of guide molecules comprising guide molecules capable of binding one or more target sequences of a target molecule and designed to form a complex with a Cas protein (para [0005] "a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind to corresponding target molecules"; [0066] "C2c2 is not sensitive to single mismatches, but can distinguish between single nucleotide differences in target when loaded with crRNAs with additional mismatches"); b. distributing a plurality of sets of guide molecules, thereby spatially segregating each set of guide molecules (para [0026] "distributing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system comprising an effector protein, one or more guide RNAs"; [0276] "Each discrete volume may comprise a different guide RNA specific for a different target molecule"); C. distributing to each set of guide molecules a sample solution, detection reagents, and a reporter construct comprising a non-target sequence, and a Cas protein (para [0026] "distributing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system comprising an effector protein, one or more guide RNAs"; [0024] "the RNA- based masking agent is an aptamer that sequesters an enzyme, wherein the enzyme generates a detectable signal upon release from the aptamer by acting upon a substrate"; [0454]-[0457] "Here we describe SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), an in vitro nucleic acid detection platform with attomolar sensitivity based on nucleic acid amplification and 3 Casl3a- mediated collateral cleavage of a commercial reporter RNA"; Note, the "reporter construct" is either (1) the enzyme/aptamer or (2) the reporter RNA); d. initiating a detection reaction, wherein the Cas protein cleaves the non-target sequence of the reporter constructs once activated by the target sequences (para [0026] "activating the CRISPR effector protein via binding of the one or more guide RNAs to the one or more target molecules, wherein activating the CRISPR effector protein results in modification of the RNA-based masking construct such that a detectable positive signal is produced"; [0047] "Upon recognition of target RNA, the collateral effect causes C2c2 to cut the cleavage reporter, generating fluorescence"; [0150]; [0155] "Binding of the one or more guide RNAs to a target nucleic acid in turn activates the CRISPR effector protein. Once activated, the CRISPR effector protein then deactivates the masking construct, for example, by cleaving the masking construct such that a detectable positive signal is unmasked, released, or generated"); and e. measuring the signal generated from the reporter construct associated with each set of guide molecules from cleavage of the non-target sequence of the reporter constructs (para [0026] "a detectable positive signal is produced; and detecting the detectable positive signal, wherein detection of the detectable positive signal indicates a presence of one or more target molecules in the sample"; [0155] "cleaving the masking construct such that a detectable positive signal is unmasked, released, or generated. Detection of the positive detectable signal in an individual discrete volume indicates the presence of the target molecules").
With regards to claim 2, Collins teaches distributing a plurality of sets of guide molecules comprises distributing the plurality of sets of guide molecules to individual discrete volumes (para [0026] "the individual discrete volumes comprising one or more guide RNAs"; [0263] "Within each defined spot, reagents of the system described herein are applied to the individual spots. Each spot may contain the same reagents except for a different guide RNA or set of guide RNAs").
With regards to claim 3, Collins teaches wherein each individual discrete volume comprises a set of one or more detection beads, wherein each detection bead comprises the set of guide molecules capable of binding the one or more target sequences of the target molecule (para [0262]-[0263] "Exemplary discrete volumes or spaces useful in the disclosed methods include droplets (for example, microfluidic droplets and/or emulsion droplets), hydrogel beads Within each defined spot, reagents of the system described herein are applied to the individual spots. Each spot may contain the same reagents except for a different guide RNA or set of guide RNAs to screen for multiple targets at once"; [0270] "A detection construct comprising a fluorescent detectable label may be cast into the droplet comprising unpolymerized gel monomer. Upon polymerization of the gel monomer to form a bead within a droplet. Because gel polymerization is through free-radical formation, the fluorescent reporter becomes covalently bound to the gel").
With regards to claim 4, Collins teaches further comprising the step of mixing the detection bead with Cas protein, thereby coupling the Cas protein to the set of guide molecules disposed on the bead (para [0026]; [0281] "the system may include a masking agent, CRISPR effector protein, and guide RNAs specific for a target molecule. Upon activation of the LOC, the microfluidic device may mix the sample and assay reagents"; [0282] "In certain embodiments, separate sensors each associated with a different CRISPR effector protein and guide RNA immobilized to a sensor are used to detect multiple target molecules").
With regards to claim 6, Collins teaches further comprising multiple sets of detection beads, each bead in a given set comprising guide molecules configured to detect a particular target molecule, and each different set of beads configured to detect a different target molecule such that detection of multiple target molecules is screened at once (para [0263] "Each spot may contain the same reagents except for a different guide RNA or set of guide RNAs, or where applicable, a different detection aptamer to screen for multiple targets at once"; [0282] "the test area can be very small allowing for more tests to be done in a given area separate sensors each associated with a different CRISPR effector protein and guide RNA immobilized to a sensor are used to detect multiple target molecules. Not being bound by a theory, activation of different sensors may be distinguished by the wireless device").
With regards to claim 7, Collins teaches wherein the reporter construct is attached to a reporter bead (para [0225]-[0226] "In certain example embodiments, the masking construct may be immobilized on a solid substrate in an individual discrete volume (defined further below) and sequesters a single reagent. For example, the reagent may be a bead comprising a dye. In certain aspects, the masking construct that binds the immobilized reagent is a RNA aptamer. The immobilized reagent may be a protein and the labeled minding partner may be a labeled antibody").
With regards to claim 8, Collins teaches wherein the reagents are encoded delivered to the individual discrete volumes (para [0026] "the individual discrete volumes comprising. one or more guide RNAs"; [0262]-[0263] "Within each defined spot, reagents of the system described herein are applied to the individual spots. Each spot may contain the same reagents except for a different guide RNA or set of guide RNAs").
With regards to claim 9, Collins teaches wherein the detection bead is encapsulated in a droplet (para [0020] "In some embodiments, the individual discrete volumes are droplets"; [0270] "A detection construct comprising a fluorescent detectable label may be cast into the droplet comprising unpolymerized gel monomer. Upon polymerization of the gel monomer to form a bead within a droplet").
With regards to claim 10, wherein a reporter bead is encapsulated in the same droplet as the detection bead, or in a separate droplet that can be fused with the droplet comprising the detection bead (para [0020] "In some embodiments, the individual discrete volumes are droplets"; [0225] "the masking construct may be immobilized on a solid substrate in an individual discrete volume (defined further below) and sequesters a single reagent. For example, the reagent may be a bead the immobilized masking agent is a RNA-based aptamer that can be cleaved by the activated effector protein upon detection of a target molecule"; [0269] "a first set of droplets may be formed containing samples to be screened and a second set of droplets formed containing the elements of the systems described herein. The first and second set of droplets are then merged and then diagnostic methods as described herein are carried out on the merged droplet set").
With regards to claim 12, Collins teaches distributing into wells (0262) Collins teaches the use of microfluidic devices (0246).
With regards to claim 14, Collins teaches “[0254] In certain example embodiments, the guide RNA may be labeled with a binding tag. In certain example embodiments, the entire guide RNA may be labeled using in vitro transcription (IVT) incorporating one or more biotinylated nucleotides, such as, biotinylated uracil. In some embodiments, biotin can be chemically or enzymatically added to the guide RNA, such as, the addition of one or more biotin groups to the 3' end of the guide RNA The binding tag may be used to pull down the guide RNA/target nucleic acid complex after binding has occurred, for example, by exposing the guide RNA/target nucleic acid to a streptavidin coated solid substrate.” Collins teaches, “some embodiments, the solid support comprise microspheres or beads. "Microspheres," "bead," "particles," are intended to mean within the context of a solid substrate to mean small discrete particles made of various material including, but not limited to, plastics, ceramics, glass, and plystyrene. In certain embodiments, the microspheres are magnetic microsphers or beads.” (0252)
The specification recites “optical barcode” 12 times. Thus the broadest reasonable interpretation is any barcode.
With regards to claim 16, Collins teaches, “ Genetically modified microbes may be modified to include a nucleic acid barcode sequence that identifies the particular genetic modification carried by a particular microbial cell or population of microbial cells. A barcode is s short sequence of nucleotides (for example, DNA, RNA, or combinations thereof) that is used as an identifier. A nucleic acid barcode may have a length of 4-100 nucleotides and be either single or double-stranded. Methods for identifying cells with barcodes are known in the art. Accordingly, guide RNAs of the CRISPR effector systems described herein may be used to detect the barcode.” (0327)
With regards to claim 17, Collins teaches magnetic microspheres or beads.
With regards to claim 18, Collins teaches, “[0245] In some embodiments, amplification reagents as described herein may be appropriate for use in hot-start amplification. Hot start amplification may be beneficial in some embodiments to reduce or eliminate dimerization of adaptor molecules or oligos, or to otherwise prevent unwanted amplification products or artifacts and obtain optimum amplification of the desired product. Many components described herein for use in amplification may also be used in hot-start amplification. In some embodiments, reagents or components appropriate for use with hot-start amplification may be used in place of one or more of the composition components as appropriate. For example, a polymerase or other reagent may be used that exhibits a desired activity at a particular temperature or other reaction condition. In some embodiments, reagents may be used that are designed or optimized for use in hot-start amplification, for example, a polymerase may be activated after transposition or after reaching a particular temperature. Such polymerases may be antibody-based or apatamerbased. Polymerases as described herein are known in the art. Examples of such reagents may include, but are not limited to, hot-start polymerases, hot-start dNTPs, and photo-caged dNTPs. Such reagents are known and available in the art. One of skill in the art will be able to determine the optimum temperatures as appropriate for individual reagents [0246] Amplification of nucleic acids may be performed using specific thermal cycle machinery or equipment, and may be performed in single reactions or in bulk, such that any desired number of reactions may be performed simultaneously. In some embodiments, amplification may be performed using microfluidic or robotic devices, or may be performed using manual alteration in temperatures to achieve the desired amplification. In some embodiments, optimization may be performed to obtain the optimum reactions conditions for the particular application or materials.” Thus Collins teaches detection with amplification.
With regards to claim 19, Collins teaches Isothermal amplification.
With regards to claim 20. Collins teaches, “While some Cas enzymes target DNA
(7, 8), single effector RNA-guided RNases, such as Casl3a” (0455)
With regards to claim 21, Collins teaches Cas13a which Type VI. (0455)
With regards to claim 23, Collins teaches, “A positive detectable signal may be any signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art. “ (0218)
The specification recites “optical barcode” 12 times. Thus the broadest reasonable interpretation is any barcode.
With regards to claim 24, Collins teaches, “ Genetically modified microbes may be modified to include a nucleic acid barcode sequence that identifies the particular genetic modification carried by a particular microbial cell or population of microbial cells. A barcode is s short sequence of nucleotides (for example, DNA, RNA, or combinations thereof) that is used as an identifier. A nucleic acid barcode may have a length of 4-100 nucleotides and be either single or double-stranded. Methods for identifying cells with barcodes are known in the art. Accordingly, guide RNAs of the CRISPR effector systems described herein may be used to detect the barcode.” (0327)
With regards to claim 25, Collins teaches freeze drying of the system. (0276).
With regards to claim 27, Collins teaches, “[0229] When the RNA bridge is cut by the activated CRISPR effector, the beforementioned color shift is observed. In certain example embodiments the particles are colloidal metals. In certain other example embodiments, the colloidal metal is a colloidal gold. In certain example embodiments, the colloidal nanoparticles are 15 nm gold nanoparticles (AuNPs). Due to the unique surface properties of colloidal gold nanoparticles, maximal absorbance is observed at 520 nm when fully dispersed in solution and appear red in color to the naked eye. Upon aggregation of AuNPs, they exhibit a red-shift in maximal absorbance and appear darker in color, eventually precipitating from solution as a dark purple aggregate. In certain example embodiments the nanoparticles are modified to include DNA linkers extending from the surface of the nanoparticle. Individual particles are linked together by single-stranded RNA (ssRNA) bridges that hybridize on each end of the RNA to at least a portion of the DNA linkers. Thus, the nanoparticles will form a web of linked particles and aggregate, appearing as a dark precipitate. Upon activation of the CRISPR effectors disclosed herein, the ssRNA bridge will be cleaved, releasing the AU NPS from the linked mesh and producing a visible red color. Example DNA linkers and RNA bridge sequences are listed below. Thiol linkers on the end of the DNA linkers may be used for surface conjugation to the AuNPS. Other forms of conjugation may be used. In certain example embodiments, two populations of AuNPs may be generated, one for each DNA linker. This will help facilitate proper binding of the ssRNA bridge with proper orientation. In certain example embodiments, a first DNA linker is conjugated by the 3' end while a second DNA linker is conjugated by the 5' end.”
With regards to claim 49-50, Collins teaches, “[0270] In certain example embodiments, the system and/or device may be adapted for conversion to a flow-cytometry readout in or allow to all of sensistive and quantitative measurements of millions of cells in a single experiment and improve upon existing flow-based methods, such as the PrimeFlow assay. “
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.
Claim(s) 1-4, 6-10, 12-14, 16-21, 23-25, 27, and 49-50 is/are rejected under 35 U.S.C. 103 as being unpatentable over Collins (WO2018170340).
With regards to claim 1, Collins teaches a high throughput method for detecting target molecules (para [0026] "the invention provides a method for detecting target RNAs in samples") comprising a. generating sets of guide molecules, each set of guide molecules comprising guide molecules capable of binding one or more target sequences of a target molecule and designed to form a complex with a Cas protein (para [0005] "a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind to corresponding target molecules"; [0066] "C2c2 is not sensitive to single mismatches, but can distinguish between single nucleotide differences in target when loaded with crRNAs with additional mismatches"); b. distributing a plurality of sets of guide molecules, thereby spatially segregating each set of guide molecules (para [0026] "distributing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system comprising an effector protein, one or more guide RNAs"; [0276] "Each discrete volume may comprise a different guide RNA specific for a different target molecule"); C. distributing to each set of guide molecules a sample solution, detection reagents, and a reporter construct comprising a non-target sequence, and a Cas protein (para [0026] "distributing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system comprising an effector protein, one or more guide RNAs"; [0024] "the RNA- based masking agent is an aptamer that sequesters an enzyme, wherein the enzyme generates a detectable signal upon release from the aptamer by acting upon a substrate"; [0454]-[0457] "Here we describe SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), an in vitro nucleic acid detection platform with attomolar sensitivity based on nucleic acid amplification and 3 Casl3a- mediated collateral cleavage of a commercial reporter RNA"; Note, the "reporter construct" is either (1) the enzyme/aptamer or (2) the reporter RNA); d. initiating a detection reaction, wherein the Cas protein cleaves the non-target sequence of the reporter constructs once activated by the target sequences (para [0026] "activating the CRISPR effector protein via binding of the one or more guide RNAs to the one or more target molecules, wherein activating the CRISPR effector protein results in modification of the RNA-based masking construct such that a detectable positive signal is produced"; [0047] "Upon recognition of target RNA, the collateral effect causes C2c2 to cut the cleavage reporter, generating fluorescence"; [0150]; [0155] "Binding of the one or more guide RNAs to a target nucleic acid in turn activates the CRISPR effector protein. Once activated, the CRISPR effector protein then deactivates the masking construct, for example, by cleaving the masking construct such that a detectable positive signal is unmasked, released, or generated"); and e. measuring the signal generated from the reporter construct associated with each set of guide molecules from cleavage of the non-target sequence of the reporter constructs (para [0026] "a detectable positive signal is produced; and detecting the detectable positive signal, wherein detection of the detectable positive signal indicates a presence of one or more target molecules in the sample"; [0155] "cleaving the masking construct such that a detectable positive signal is unmasked, released, or generated. Detection of the positive detectable signal in an individual discrete volume indicates the presence of the target molecules").
Collins does not specifically teach beads are sized between 2 micrometers and 100 micrometers.
MPEP 2144.05 III states:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.). For more recent cases applying this principle, see Merck & Co. Inc. v. Biocraft Laboratories Inc., 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989); In re Kulling, 897 F.2d 1147, 14 USPQ2d 1056 (Fed. Cir. 1990); and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997).
Similarly, a prima facie case of obviousness exists where the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have the same properties. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985) (Court held as proper a rejection of a claim directed to an alloy of “having 0.8% nickel, 0.3% molybdenum, up to 0.1% iron, balance titanium” as obvious over a reference disclosing alloys of 0.75% nickel, 0.25% molybdenum, balance titanium and 0.94% nickel, 0.31% molybdenum, balance titanium.).
Collins teaches, “The bead sizes range from nanometers, e.g. 100 nm, to millimeters, e.g. 1 mm.” (0251).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use beads of 2 micrometers and 100 micrometers. The artisan would be motivated as Collins suggest ranges which encompass those claimed and would have expected them to have the same properties as those claimed. The artisan would have a reasonable expectation of success as the artisan is merely using known beads.
With regards to claim 2, Collins teaches distributing a plurality of sets of guide molecules comprises distributing the plurality of sets of guide molecules to individual discrete volumes (para [0026] "the individual discrete volumes comprising one or more guide RNAs"; [0263] "Within each defined spot, reagents of the system described herein are applied to the individual spots. Each spot may contain the same reagents except for a different guide RNA or set of guide RNAs").
With regards to claim 3, Collins teaches wherein each individual discrete volume comprises a set of one or more detection beads, wherein each detection bead comprises the set of guide molecules capable of binding the one or more target sequences of the target molecule (para [0262]-[0263] "Exemplary discrete volumes or spaces useful in the disclosed methods include droplets (for example, microfluidic droplets and/or emulsion droplets), hydrogel beads Within each defined spot, reagents of the system described herein are applied to the individual spots. Each spot may contain the same reagents except for a different guide RNA or set of guide RNAs to screen for multiple targets at once"; [0270] "A detection construct comprising a fluorescent detectable label may be cast into the droplet comprising unpolymerized gel monomer. Upon polymerization of the gel monomer to form a bead within a droplet. Because gel polymerization is through free-radical formation, the fluorescent reporter becomes covalently bound to the gel").
With regards to claim 4, Collins teaches further comprising the step of mixing the detection bead with Cas protein, thereby coupling the Cas protein to the set of guide molecules disposed on the bead (para [0026]; [0281] "the system may include a masking agent, CRISPR effector protein, and guide RNAs specific for a target molecule. Upon activation of the LOC, the microfluidic device may mix the sample and assay reagents"; [0282] "In certain embodiments, separate sensors each associated with a different CRISPR effector protein and guide RNA immobilized to a sensor are used to detect multiple target molecules").
With regards to claim 6, Collins teaches further comprising multiple sets of detection beads, each bead in a given set comprising guide molecules configured to detect a particular target molecule, and each different set of beads configured to detect a different target molecule such that detection of multiple target molecules is screened at once (para [0263] "Each spot may contain the same reagents except for a different guide RNA or set of guide RNAs, or where applicable, a different detection aptamer to screen for multiple targets at once"; [0282] "the test area can be very small allowing for more tests to be done in a given area separate sensors each associated with a different CRISPR effector protein and guide RNA immobilized to a sensor are used to detect multiple target molecules. Not being bound by a theory, activation of different sensors may be distinguished by the wireless device").
With regards to claim 7, Collins teaches wherein the reporter construct is attached to a reporter bead (para [0225]-[0226] "In certain example embodiments, the masking construct may be immobilized on a solid substrate in an individual discrete volume (defined further below) and sequesters a single reagent. For example, the reagent may be a bead comprising a dye. In certain aspects, the masking construct that binds the immobilized reagent is a RNA aptamer. The immobilized reagent may be a protein and the labeled minding partner may be a labeled antibody").
With regards to claim 8, Collins teaches wherein the reagents are encoded delivered to the individual discrete volumes (para [0026] "the individual discrete volumes comprising. one or more guide RNAs"; [0262]-[0263] "Within each defined spot, reagents of the system described herein are applied to the individual spots. Each spot may contain the same reagents except for a different guide RNA or set of guide RNAs").
With regards to claim 9, Collins teaches wherein the detection bead is encapsulated in a droplet (para [0020] "In some embodiments, the individual discrete volumes are droplets"; [0270] "A detection construct comprising a fluorescent detectable label may be cast into the droplet comprising unpolymerized gel monomer. Upon polymerization of the gel monomer to form a bead within a droplet").
With regards to claim 10, wherein a reporter bead is encapsulated in the same droplet as the detection bead, or in a separate droplet that can be fused with the droplet comprising the detection bead (para [0020] "In some embodiments, the individual discrete volumes are droplets"; [0225] "the masking construct may be immobilized on a solid substrate in an individual discrete volume (defined further below) and sequesters a single reagent. For example, the reagent may be a bead the immobilized masking agent is a RNA-based aptamer that can be cleaved by the activated effector protein upon detection of a target molecule"; [0269] "a first set of droplets may be formed containing samples to be screened and a second set of droplets formed containing the elements of the systems described herein. The first and second set of droplets are then merged and then diagnostic methods as described herein are carried out on the merged droplet set").
With regards to claim 12, Collins teaches distributing into wells (0262) Collins teaches the use of microfluidic devices (0246).
With regards to claim 14, Collins teaches “[0254] In certain example embodiments, the guide RNA may be labeled with a binding tag. In certain example embodiments, the entire guide RNA may be labeled using in vitro transcription (IVT) incorporating one or more biotinylated nucleotides, such as, biotinylated uracil. In some embodiments, biotin can be chemically or enzymatically added to the guide RNA, such as, the addition of one or more biotin groups to the 3' end of the guide RNA The binding tag may be used to pull down the guide RNA/target nucleic acid complex after binding has occurred, for example, by exposing the guide RNA/target nucleic acid to a streptavidin coated solid substrate.” Collins teaches, “some embodiments, the solid support comprise microspheres or beads. "Microspheres," "bead," "particles," are intended to mean within the context of a solid substrate to mean small discrete particles made of various material including, but not limited to, plastics, ceramics, glass, and plystyrene. In certain embodiments, the microspheres are magnetic microsphers or beads.” (0252)
The specification recites “optical barcode” 12 times. Thus the broadest reasonable interpretation is any barcode.
With regards to claim 16, Collins teaches, “ Genetically modified microbes may be modified to include a nucleic acid barcode sequence that identifies the particular genetic modification carried by a particular microbial cell or population of microbial cells. A barcode is s short sequence of nucleotides (for example, DNA, RNA, or combinations thereof) that is used as an identifier. A nucleic acid barcode may have a length of 4-100 nucleotides and be either single or double-stranded. Methods for identifying cells with barcodes are known in the art. Accordingly, guide RNAs of the CRISPR effector systems described herein may be used to detect the barcode.” (0327)
With regards to claim 17, Collins teaches magnetic microspheres or beads.
With regards to claim 18, Collins teaches, “[0245] In some embodiments, amplification reagents as described herein may be appropriate for use in hot-start amplification. Hot start amplification may be beneficial in some embodiments to reduce or eliminate dimerization of adaptor molecules or oligos, or to otherwise prevent unwanted amplification products or artifacts and obtain optimum amplification of the desired product. Many components described herein for use in amplification may also be used in hot-start amplification. In some embodiments, reagents or components appropriate for use with hot-start amplification may be used in place of one or more of the composition components as appropriate. For example, a polymerase or other reagent may be used that exhibits a desired activity at a particular temperature or other reaction condition. In some embodiments, reagents may be used that are designed or optimized for use in hot-start amplification, for example, a polymerase may be activated after transposition or after reaching a particular temperature. Such polymerases may be antibody-based or apatamerbased. Polymerases as described herein are known in the art. Examples of such reagents may include, but are not limited to, hot-start polymerases, hot-start dNTPs, and photo-caged dNTPs. Such reagents are known and available in the art. One of skill in the art will be able to determine the optimum temperatures as appropriate for individual reagents [0246] Amplification of nucleic acids may be performed using specific thermal cycle machinery or equipment, and may be performed in single reactions or in bulk, such that any desired number of reactions may be performed simultaneously. In some embodiments, amplification may be performed using microfluidic or robotic devices, or may be performed using manual alteration in temperatures to achieve the desired amplification. In some embodiments, optimization may be performed to obtain the optimum reactions conditions for the particular application or materials.” Thus Collins teaches detection with amplification.
With regards to claim 19, Collins teaches Isothermal amplification.
With regards to claim 20. Collins teaches, “While some Cas enzymes target DNA
(7, 8), single effector RNA-guided RNases, such as Casl3a” (0455)
With regards to claim 21, Collins teaches Cas13a which Type VI. (0455)
With regards to claim 23, Collins teaches, “A positive detectable signal may be any signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art. “ (0218)
The specification recites “optical barcode” 12 times. Thus the broadest reasonable interpretation is any barcode.
With regards to claim 24, Collins teaches, “ Genetically modified microbes may be modified to include a nucleic acid barcode sequence that identifies the particular genetic modification carried by a particular microbial cell or population of microbial cells. A barcode is s short sequence of nucleotides (for example, DNA, RNA, or combinations thereof) that is used as an identifier. A nucleic acid barcode may have a length of 4-100 nucleotides and be either single or double-stranded. Methods for identifying cells with barcodes are known in the art. Accordingly, guide RNAs of the CRISPR effector systems described herein may be used to detect the barcode.” (0327)
With regards to claim 25, Collins teaches freeze drying of the system. (0276).
With regards to claim 27, Collins teaches, “[0229] When the RNA bridge is cut by the activated CRISPR effector, the beforementioned color shift is observed. In certain example embodiments the particles are colloidal metals. In certain other example embodiments, the colloidal metal is a colloidal gold. In certain example embodiments, the colloidal nanoparticles are 15 nm gold nanoparticles (AuNPs). Due to the unique surface properties of colloidal gold nanoparticles, maximal absorbance is observed at 520 nm when fully dispersed in solution and appear red in color to the naked eye. Upon aggregation of AuNPs, they exhibit a red-shift in maximal absorbance and appear darker in color, eventually precipitating from solution as a dark purple aggregate. In certain example embodiments the nanoparticles are modified to include DNA linkers extending from the surface of the nanoparticle. Individual particles are linked together by single-stranded RNA (ssRNA) bridges that hybridize on each end of the RNA to at least a portion of the DNA linkers. Thus, the nanoparticles will form a web of linked particles and aggregate, appearing as a dark precipitate. Upon activation of the CRISPR effectors disclosed herein, the ssRNA bridge will be cleaved, releasing the AU NPS from the linked mesh and producing a visible red color. Example DNA linkers and RNA bridge sequences are listed below. Thiol linkers on the end of the DNA linkers may be used for surface conjugation to the AuNPS. Other forms of conjugation may be used. In certain example embodiments, two populations of AuNPs may be generated, one for each DNA linker. This will help facilitate proper binding of the ssRNA bridge with proper orientation. In certain example embodiments, a first DNA linker is conjugated by the 3' end while a second DNA linker is conjugated by the 5' end.”
With regards to claim 49-50, Collins teaches, “[0270] In certain example embodiments, the system and/or device may be adapted for conversion to a flow-cytometry readout in or allow to all of sensistive and quantitative measurements of millions of cells in a single experiment and improve upon existing flow-based methods, such as the PrimeFlow assay. “.
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.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 1-4, 6-10, 12-14, 16-21, 23-25, 27, and 49-50 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 26-64 of copending Application No. 18235760. Although the claims at issue are not identical, they are not patentably distinct from each other because they are coextensive in scope.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
The instant claims are drawn to A high throughput method for detecting target molecules comprising a. generating sets of guide molecules, each set of guide molecules comprising guide molecules capable of binding one or more target sequences of a target molecule and designed to form a complex with a Cas protein;b. distributing a plurality of sets of guide molecules, thereby spatially segregating each set of guide molecules;c. distributing to each set of guide molecules a sample solution, detection reagents,and a reporter construct comprising a non-target sequence, and a Cas protein;d. initiating a detection reaction, wherein the Cas protein cleaves the non-target sequence of the reporter constructs once activated by the target sequences; ande. measuring the signal generated from the reporter construct associated with each set of guide molecules from cleavage of the non-target sequence of the reporter constructs.
The claims of 760 are drawn to A method of detecting target nucleic acids in one or more samples, the method comprising: contacting the one or more samples with a Type VI Cas polypeptide and a guide molecule, wherein the guide molecule is capable of forming a complex with a Type VI Cas polypeptide and directing the binding of the complex to a target nucleic acid, wherein the Type VI Cas polypeptide exhibits collateral RNAse activity upon binding to the target nucleic acid; and detecting degradation of non-target nucleic acids indicates the presence of the target nucleic acid in the sample.
Thus it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims of 790 encompass the instant claims as the contacting step of the claims of 790 at least render obvious the distributing step. Thus the claims of 790 encompass the instant invention.
Dependent claims are obvious as they coextensive in scope.
Summary
No claims are allowed.
Conclusion
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/Steven Pohnert/ Primary Examiner, Art Unit 1683
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