DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections
35 USC § 102
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.
Claims 1-6, 8, 14, 18, 20, 22, 23, 25, 27, 32, 33, 36, 37, 40, and 49 are rejected under 35 U.S.C. 102(a)1 and 102(a)(2) as being anticipated by Abudayyeh [US 20180340219 A1].
Regarding claim 1, Abudayyeh discloses a method of enriching a sample for a target RNA or DNA prior to detection by a CRISPR effector system. This method utilizes an apparatus in which a dead Cas protein is immobilized to a solid substrate and complexed with a gRNA, thus indirectly immobilizing the gRNA to the substrate. The gRNA has sufficient complementarity with a target nucleic acid to permit hybridization to occur.
A sample containing target nucleic acids is exposed to the apparatus resulting in the capture (i.e., hybridization) of the target by the CRISPR effector system, indicating conditions suitable for hybridization were used. Multiple gRNAs specific to different target nucleic acids can be used in a single assay, allowing for the detection of multiple targets and/or multiple variants of a single target. Once the target is captured, non-target (i.e., unhybridized nucleic acids) are washed away and the target is released from the system for further detection by methods additionally described by Abudayyeh [Abudayyeh, 0201, 0260-0261].
Regarding claims 2-4, Abudayyeh’s disclosed methods can be applied to a number of applications including the detection of target nucleic acids in both clinical and environmental samples, as well as for pathogen detection in said samples [Abudayyeh, 0274-0277, 0296].
Regarding claims 5 and 6, Abudayyeh’s method can enrich a sample for target RNA or DNA prior to its detection [Abudayyeh, 0201, 0257].
Regarding claim 18, the solid substrate is described as a planar structure, such as an array, comprised of multiple discrete volumes [Abudayyeh, 0260, 0272].
Regarding claims 22, 23, and 25, Abudayyeh describes a “CRISPR effector protein/guide RNA complex” as being used in this enrichment method wherein the CRISPR protein is dead. The effector protein can be any number of Cas proteins, including Cas9 and Cas13a/b [Abudayyeh, 0169-0173, 0261].
Regarding claim 20, the gRNA molecules can comprise non-naturally occurring nucleic acids, nucleotides, nucleotide analogs, and/or chemical modifications [Abudayyeh, 0209].
Regarding claim 32, 33, 36, 37, Abudayyeh discloses a solid substrate comprising multiple discrete volumes, each comprising a dead Cas protein and a guide RNA (gRNA). The gRNA has sufficient complementarity with a target nucleic acid to permit hybridization to occur, resulting in sequence-specific binding by the CRISPR effector system. The CRISPR protein is immobilized to the solid substrate, thus indirectly immobilizing the gRNA to the substrate surface. The solid substrate is described as a planar structure, such as an array, comprised of a variety of materials such as glass, plastics, polysaccharides, nylon or nitro cellulose, ceramics, resins, silica or silicon-based materials, carbon and metals [Abudayyeh, 0020, 0172, 0201, 0260, 0272].
Regarding claim 40, Abudayyeh teaches that kits and systems for using their disclosed methods can be designed to be useable in the field so that diagnostics can be performed onsite rather than requiring sending samples to another part of the country or the world thus shortening the time between sample collection and obtainment of results [Abudayyeh, 0326]. As Abudayyeh teaches a microarray with the claimed features, it can be assumed that this array would be present in a kit produced from their teachings.
Claims 32, 33, 36, and 37 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Lucas [US 20260043071 A1, Effective Filing Date: 06/07/2023].
Lucas’s describes a method for detecting a target nucleic acid which utilizes a gRNA immobilized to a surface, such as a microarray, and complexed with a Cas enzyme. Therefore, Lucas also teaches the microarray itself which may be printed onto a functionalized glass surface. This microarray can be designed to detect multiple targets such that each spot on the microarray surface represents a specific Cas/gRNA combination, demonstrating the immobilization of a plurality of gRNA molecules on the microarray. Target capture occurs as a result of complementarity between a gRNA molecule and a target nucleic acid. The Cas used in the complex may be enzymatically active or dead [Lucas, 0005, 0016-0017, 0039-0040, 0075-0076, 0090].
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 40 is rejected under 35 U.S.C. 103 as being unpatentable over Lucas in view of Polansky [US 20040023207 A1].
As previously described, Lucas teaches a microarray comprising a plurality of gRNA molecules immobilized to its surface and complexed with a Cas enzyme so that each spot on the microarray represents a specific Cas/gRNA combination wherein the gRNA molecules are complementary to a target nucleic acid [Lucas, 0005, 0016, 0075-0076]. Lucas does not teach including this microarray in a kit.
However, Polanksy teaches that the inclusion of individual components in a commercial kit imparts well known advantages such as convenience and reproducibility due to manufacturing standardization, quality control, and validation procedures [Polanksy, 0919]. Therefore, one of ordinary skill in the art prior to the effective filling date of the claimed invention would have been motivated to include Lucas’ microarray in a kit thereby achieving the well-known benefits described by Polansky.
Claims 1-6, 8, 18, 20, 22, 23, 25, 27 are rejected under 35 U.S.C. 103 as being unpatentable over Lucas in view of Eads [Eads B, et al. Methods in enzymology. 2006 Jan 1;411:34-49].
Regarding claim 1, Lucas describes a method for detecting a target nucleic acid. First, a guide RNA (gRNA) is immobilized to a surface, such as a microarray, and complexed with a Cas enzyme. Once complexed, a target nucleic acid, which was isolated from a sample, is added to the microarray, captured via a complementary portion of the gRNA, and detected via labeling of the target nucleic acid either prior to or after capture. As capture occurs as a result of hybridization between complementary regions of the gRNA and the target, it can be assumed that the conditions are suitable for this to occur. Lucas further teaches that this method can be designed to allow for multiplexing such that each spot on the multiplex represents a specific Cas/gRNA combination. This further demonstrates that Lucas immobilizes a plurality of gRNA molecules to the array surface [Lucas, 0005, 0016-0021, 0075-0077, 0082].
Lucas does not teach washing the apparatus (i.e., microarray) to remove unhybridized nucleic acids. However, Eads describes problems commonly encountered when performing microarray experiments and various methods for avoiding or correcting them. For example, “comet tailing” and increased background fluorescence can be caused by inadequate washing which may be avoided by empirically determining the optimal wash parameters for a specific platform and/or sample [Eads, abstract, 38, 42-44].
Therefore, one of ordinary skill in the art prior to the effective filing date of the claimed invention would have been motivated to incorporate a post-hybridization washing step into Lucas’ method in order to avoid non-specific hybridization of nucleic acids as Eads teaches that this can lead to visualization problems which are known to occur in microarray experiments (i.e., comet tailing and high background fluorescence). Thus, incorporating Eads’ post-hybridization washing technique into Lucas’ microarray-based detection method would have amounted to the use of a known technique to improve similar devices (methods or products) in the same way, namely the reduction of nonspecific binding and reduction of background fluorescence, and is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, C.).
Regarding claims 2-4, the target nucleic acid may be isolated from a pathogen, a mosquito, or a sample from a subject including blood, urine, bone marrow, serum, saliva, semen, cerebrospinal fluid, oral fluid, stool, sputum, and tissue [Lucas, 0018-0021].
Regarding claims 5 and 6, the target nucleic acid can be DNA or RNA [Lucas, 0079].
Regarding claim 8, multiple embodiments for labeling the target nucleic acid are provided. For example, the target nucleic acid can be labeled with a labeling dye which is conjugated with a fluorophore, or with biotin which reacts with a streptavidin-conjugated quantum dot [Lucas, 0112-0115].
Regarding claim 18, multiple options exists for the immobilization surface including a microarray. This format allows for hundreds of assays to be multiplexed together with each spot representing a specific Cas/gRNA combination that is individually distinguishable [Lucas, 0016, 0082, 0085].
Regarding claim 20, the immobilized gRNA can be modified to facilitate attachment to the array. For example, the gRNA may be amine-conjugated to allow attachment to a carboxylated surface via EDC-NHS chemistry [Lucas, 0007].
Regarding claims 22, 23, and 25, as previously discussed, the gRNA is complexed with a Cas enzyme. This Cas may be an enzymatically active or dead version of Cas9, Cas12, Cas13, or Cas14, or one of their subtypes [Lucas, 0005, 0039-0040, 0090].
Regarding claim 27, Eads describes the use of a blocking agent during pre-hybridization to prevent nonspecific binding as an additional method for avoiding visualization problems common to microarrays, such as black holes and increased background fluorescence [Eads, p42].
Claims 14 and 49 are rejected under 35 U.S.C. 103 as being unpatentable over Lucas and Eads, as applied to claim 1 above, and further in view of Xu [Xu X, et al. bioRxiv. 2020 May 14: p1-23].
Lucas is applied to the relevant teachings of claim 1 as discussed above and is incorporated herein by reference. Lucas further teaches that immobilization can occur through a variety of methods including covalent binding and that the gRNA is complexed with either a dead or enzymatically active Cas enzyme [Lucas, 0089-0090]. Lucas does not teach that the target nucleic acid is present in the sample at a concentration less than 1nM or that the sample is contacted with the microarray for less than 24 hours.
Xu developed a CRISPR/Cas9-assisted DNA detection (CADD) method in which a pair of gRNAs, each coupled to a dCas9 enzyme, are designed to capture a different portion of a target DNA and contain a short extended 3` terminal capture sequence that can anneal with other functional oligonucleotides. The pair will bind to their target DNA and then be captured onto the surface of a bead or microwell through complementary annealing between the capture sequence of the first gRNA and a surface bound oligonucleotide. Once bound, the capture sequence of the second gRNA anneals to a biotinylated oligonucleotide which facilitates detection via a horseradish peroxidase/TMB process. This method was applied to HPV detection and successfully detected HPV in plasmid samples (bead and plate-based) and clinical samples (plate based) in which the target was present at concentrations down to 100aM. The entire detection process can be finished in 30 minutes demonstrating its potential for rapid on-the-spot/point-of-care detection [Xu, p6-9, Figure 5].
A person of ordinary skill in the art prior to the effective filing date of the claimed invention would have recognized that Lucas and Xu both teach the use of sequence-specific gRNA/Cas complexes for the capture and detection of target nucleic acids on solid supports without the use of amplification methods. Xu further demonstrates that target capture using a system similar to Lucas can be performed at sub-nM concentrations and in as little as 30 minutes. Lucas additionally states that a benefit of their system is its ability to be configured to function with a very rapid time to result and can be reconfigured to accommodate detection of new pathogens and/or sequence variants within 24 hours of discovery. Therefore, the skilled artisan would have had a reasonable expectation that Lucas’s method could have been successfully performed with similar time and concentration constraints as Xu and would have been motivated to do so in order to achieve a simple, rapid, sensitive, and highly multiplexable method which is not reliant on amplification procedures and is capable of adapting to the needs of the artisan [Lucas, 0073, 0083-0087].
Conclusion
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/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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