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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 2/11/2026 has been entered.
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claims 30-34, 41, and 44-45 remain withdrawn. Claims 1-2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-57, and 59-63 are pending and are examined on the merits herein.
As noted in the Final Rejection mailed 11/20/2025, Applicant has canceled claim 19 in the Remarks provided on 7/24/2025, but this claim does appear as both pending and canceled in the claims submitted 2/11/2026. It will be considered canceled for the purposes of examination, as a canceled claim may only be reinstated by a subsequent amendment presenting the claim as a new claim with a new claim number. See MPEP 608.01(s).
Thus, though Applicant has stated that claim 19 is pending in this application in their most recent Remarks submitted 2/11/2026, this is not considered accurate, and claim 19 is not examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 2/11/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Response to Applicant’s Amendments
Nucleotide and/or Amino Acid Sequence Disclosure Objections
The specification was objected to because the Sequence Incorporation by Reference paragraph was directed to a previous version of the Sequence Listing. In light of Applicant’s amendments to the specification submitted 2/11/2026, this objection has been withdrawn.
Claim Objections
Claims 1 and 59 were objected to due to minor informalities. In light of Applicant’s amendments to the claims submitted 2/11/2026, these objections have been withdrawn, but see new grounds of objection below.
35 USC 103 Rejections
Claims 1, 2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-55, 57, 61, and 63 are rejected under 35 U.S.C. 103 as being unpatentable over Zheng et al. (WO 2017/070056), in view of Blainey et al. (WO 2016/149661), in view of East-Seletsky et al. (Nature, 2016), and in view of Gootenberg et al. (Science, 2017).
Claims 59-60 and 62 are rejected under 35 U.S.C. 103 as being unpatentable over Zheng et al. (WO 2017/070056) in view of Blainey et al. (WO 2016/149661) and in view of East-Seletsky et al. (Nature, 2016).
Claim 56 is rejected under 35 U.S.C. 103 as being unpatentable over Zheng et al. (WO 2017/070056), in view of Blainey et al. (WO 2016/149661), in view of East-Seletsky et al. (Nature, 2016), in view of Gootenberg et al. (Science, 2017), and further in view of Jabado et al. (Nucleic Acids Research, 2008).
In light of Applicant’s amendments to the claims submitted 2/11/2026, these rejections have been withdrawn, but see “Response to Applicant’s Arguments” and new grounds of rejection below.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections presented in the Final Rejection mailed 11/20/2025, Applicant argues that the cited combination of references fails to teach the claimed two-droplet-set architecture, which enables multiplexed detection. Particularly, Applicant argues that Zheng, the primary reference used, does not teach maintaining distinct first and second droplets until capture in the microwells. Applicant states that this particular setup allows for unprecedented multiplexing (Remarks, pages 11-12). In combining Zheng and Blainey, Applicant argues that their combination lacks articulated reasoning, that the references teach away from their combination, and that the combination uses the claimed invention as a roadmap, thus relying on impermissible hindsight (Remarks, pages 12-13). Specifically, Applicant states that Zheng would have no reason to add optical barcodes to track droplet content, and the results of combining this with Blainey would not predictably result in the claimed invention. East-Seletsky also does not allegedly remedy these deficiencies (Remarks, pages 13-14). Specifically regarding the rejection of claim 1 and its dependents, the Examiner has allegedly provided no proper reasoning to incorporate the teachings of Gootenberg into the rejection, as Gootenberg is not drawn to droplet-based microwell arrays.
Firstly, regarding Applicant’s arguments concerning the multiplexing ability of the invention, though the method of instant claim 1 can be used to simultaneously detect multiple targets from a large plurality of samples, the number of samples used could be as few as two. This means that the second set of droplets could, in total, contain isolated or extracted nucleic acid from as few as two targets. For the first droplets, a minimum of one gRNA is required to hybridize to one target nucleotide sequence from one sample. Thus, the first droplets can also contain, in total, a minimum of two different CRISPR systems, one for each sample. It is noted that in the preamble, it is stated “detecting simultaneously multiple target nucleotide sequences in nucleic acid isolates or extracts of a plurality of samples.” These “multiple target nucleic sequences” encompasses multiple sequences total – e.g. one sequence from each of multiple samples – as well as multiple sequences for each of the multiple samples. So, while it is possible to perform large-scale multiplexing with the claimed invention, such multiplexing is not required. Furthermore, when Applicant describes the “unprecedent multiplexing” of their invention in their Remarks, they are referring to particular working examples presented in paras. 573 and 596 of the published application that are not currently commensurate in scope with the claimed invention. The results of the use of the claimed invention are also not stated to be unexpected, nor is this “unprecedented multiplexing” argued to fulfill a long-felt need in the art, solve a problem others have failed to solve, or involve any other potential secondary considerations. Therefore, the fact that the claimed invention can be used for large-scale multiplexing that may not be explicitly discussed in the prior art references provided does not in itself obviate the existing obviousness rejections. If Applicant wishes to have the claimed invention further considered with regard to unexpected results and/or other secondary considerations, they are directed to MPEP 716.
In instant claim 59, samples are not discussed until the final wherein clause (though see the 35 USC 112(b) Rejections below), where “one of the samples” is recited. This also means that as few as two samples may be used. The second droplets do not specify that the nucleic acid isolates/extracts must be from the samples, and the first droplets must each hybridize to at least one target nucleotide of a sample. Thus, while it is possible to use a large amount of samples in this claim, it is also not required, in a manner similar to that of claim 1. Therefore, the fact that the claimed invention can be used for large-scale multiplexing that may not be explicitly discussed in the prior art references provided does not in itself obviate the existing obviousness rejections, as is the case for instant claim 1.
Secondly, regarding the teachings of Zheng in the context of Applicant’s arguments, Zheng teaches droplets containing beads with CRISPR systems encapsulated in droplets (thus making these droplets analogous to the first droplets), as well as droplets containing lysed cell materials (thus making these droplets analogous to the second droplets), where multiple organisms and cell types can be used (thus being analogous to multiple samples). See para. 31 of the Final Rejection. In paras. 100-101 and Figure 1 of Zheng, co-partitioning is discussed in the channel of a microfluidic device, where the cells and beads are pooled. Blainey does specifically teach the pooling of multiple sets of microdroplets into microwells on an array, where merging of the droplets specifically does not occur until they are present in said microwells (see para. 33 of the Final Rejection and para. 5 of Blainey, “The droplets are distributed via a flow channel running beneath the array of microwells such that droplets are able to rise out of a carrier oil via buoyancy and into an available microwell space. The droplets are then merged in parallel in each microwell to form single merged droplets comprising the combination of molecular species previously contained in each individual droplet”). In Figure 2 of Blainey, two droplets are shown in a partition as separate entities, showing that they may exist as such without immediately merging. Blainey also goes on to state, “Prior to loading the droplets onto the microfluidic device comprising an array of microwells, the droplets may be barcoded using an optical barcode. The optical barcode identifies the molecular species contained in each drop,” (para. 5). Para. 53 and Figure 7 show imaging of the molecular species in each well via use of the optical barcode, where multiple optical barcodes can be read (see also para. 14 for a description of Figure 7). Thus, these references teach encapsulation of CRISPR systems and nucleic acids in droplets, optical barcodes in individual droplets, and droplet merging of previously separate entities in partitions. In combining Zheng and Blainey in the Final Rejection, multiple motivations are provided. It is also noted that additional motivation/rationale is provided below in the 35 USC 103 Rejections to specifically address the newly amended requirement in claim 1 that the first and second optical barcodes be detected in the separate entity droplets prior to merging utilizing additional teachings from Blainey (see para. 69 below).
In considering the motivations provided in the Final Rejection, para. 34 of the Final Rejection states, “Zheng’s methods mainly concern allowing nucleic acid manipulation reagents (i.e. CRISPR systems) into partitions for particular targets, and notes that their methods can be used in screening processes (Abstract and para. 3). Blainey teaches such screening processes, and notes that their method is more robust and easily scalable compared to similar methods (Abstract and para. 2). By combining these methods, it would allow the ordinary artisan to image the target nucleic acids, which would aid in determining if particular mutants are present in a sample, or if desired genetic manipulations have occurred. Blainey also specifically teaches that targets may have optical barcodes, and incorporating these and detecting them in the method of Zheng would allow for increased accuracy in the detection of droplet contents, and would give the ordinary artisan the ability to note if a target nucleic acid, a CRISPR system, or both are present in a merged droplet.”
This provides more than a simple statement that combining aspects of Zheng and Blainey would lead to “increased accuracy” with no additional rationale. This statement of rejection notes commonalities between the two references, which supports the notion that the ordinary artisan would consider their use together, points out advantages explained by Blainey, and provides concrete reasoning in wanting to include optical barcodes with target nucleic acids (i.e. specific imaging of targets, noting of mutants or desired genetic manipulations, and the ability to optically determine if pooled droplets contain both CRISPR systems and particular targets).
Regarding further motivation and a reasonable expectation of success, para. 34 of the Final Rejection also states, “MPEP 2143 I (A) states, “The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art.” Droplet and CRISPR systems, optical barcodes, and microwell imaging are well-known in the art, as evidenced by Zheng and Blainey, and this combination would simply provide droplets with specific contents to merge and image as taught by Blainey, without changing the overall methodology. Therefore, these results would be predictable.” These rationale are not directly addressed by Applicant, and as noted above, predictability regarding performing large-scale multiplexing is not currently required by the claim.
Applicant states that this combination of references is based on impermissible hindsight. MPEP 2145 X (A) states, “Applicants may argue that the examiner’s conclusion of obviousness is based on improper hindsight reasoning. However, "[a]ny judgment on obviousness is in a sense necessarily a reconstruction based on hindsight reasoning, but so long as it takes into account only knowledge which was within the level of ordinary skill in the art at the time the claimed invention was made and does not include knowledge gleaned only from applicant’s disclosure, such a reconstruction is proper." In re McLaughlin, 443 F.2d 1392, 1395, 170 USPQ 209, 212 (CCPA 1971)…Applicants may also argue that the combination of two or more references is "hindsight" because "express" motivation to combine the references is lacking. However, there is no requirement that an "express, written motivation to combine must appear in prior art references before a finding of obviousness." Ruiz v. A.B. Chance Co., 357 F.3d 1270, 1276, 69 USPQ2d 1686, 1690 (Fed. Cir. 2004).” The Examiner does not reference Applicant’s specification in this statement of rejection, and Applicant has not pointed out where a supposed reference has occurred. And although the totality of the motivations provided by the Examiner are not explicitly stated in Zheng or Blainey, this alone does not mean the rejection is improper or relies on impermissible hindsight.
Additionally, MPEP 2141.03 I states, “"A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 421, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396.” The Examiner argues that the ordinary artisan would be capable of utilizing the teachings of Zheng and Blainey, which both rely on droplet methodologies and recite most of the limitations of the independent claims, along with ordinary creativity and knowledge available to one of ordinary skill in the art, to arrive at the claimed invention (when combined with the additional secondary references recited below).
As to the references teaching away from the claimed invention, MPEP 2143.01 I states, “The court stated that "the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed.” Zheng and Blainey separately provide inventions that do not perfectly coincide with the claimed invention, but do not directly criticize or discourage the claimed invention, and so these references are not considered to teach away from the claimed invention.
Finally, regarding the use of Gootenberg, the fact that the reference is not directly related to droplet-based microwell arrays does not mean that the reference cannot be used in an obviousness rejection. MPEP 2141.01(a) I states that in order for a reference to be used in an obviousness rejection, it must be analogous to the claimed invention. Specifically, “A reference is analogous art to the claimed invention if: (1) the reference is from the same field of endeavor as the claimed invention (even if it addresses a different problem); or (2) the reference is reasonably pertinent to the problem faced by the inventor (even if it is not in the same field of endeavor as the claimed invention).” As Gootenberg deals with detection related to CRISPR systems, it is considered to be analogous art to the claimed invention. In utilizing the teachings of Gootenberg in the rejection of claim 1 and its dependents, motivation was provided (utilizing the benefits and uses described by Gootenberg), and the results of such a combination were determined to be predictable based on the rationale of MPEP 2143 I (A), providing further motivation and a reasonable expectation of success. See para. 50 of the Final Rejection. Thus, the inclusion of this reference is considered proper, and its continued use in the 35 USC 103 Rejections is provided below.
Thus, Applicant’s arguments are overall not considered persuasive. The relevant portions of the 35 USC 103 Rejections presented in the Final Rejection are reiterated below. New grounds of rejection are provided for claims 59-60 and 62 in order to address Applicant’s amendments to the claims. The rejection of claims 1, 2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-57, 61, and 63 is also considered new grounds of rejection, as additional teachings and rationale were added to clarify the invention presented by the combination of references and address Applicant’s claim amendments.
Claim Interpretation
Regarding the use of the phrase “separate entities,” in the first newly amended wherein clause of instant claim 1, it is noted that this term does not appear in the instant specification. Para. 485 states that different species of droplet can be introduced in a microfluidic device from separate inlet microfluidic channels, and Figure 1 shows utilizing separate channels for different droplets. However, in the instant claim, the first and second droplets are pooled, captured in microwells, and then merged. The different species of droplets alone are not required to be separate entities, but all of the individual droplets themselves are considered separate. “Separate entities” will be interpreted in the context of the claim to simply mean that the droplets each exist on their own, without being merged with another droplet. It is noted in claim 1 that the droplets must exist as separate entities during the initial detection of the first and second optical barcodes.
This interpretation also applies to the use of “separate entities” in instant claim 59, though in this claim, only detection of the signals in merged droplets is required.
Claim Objections
Claim 1 is objected to because of the following informality: in the second phrase of the claim after the preamble, it is recommend to have “of the first set” read “of the first set of droplets” to better match the language used in the next phrase with regard to the second droplets (“of the second set of droplets”). Appropriate correction is required.
Claim 2 is objected to because of the following informality: for consistency of format, it is recommended that when listing PCR in the list of potential amplifying methods, the claim state “polymerase chain reaction (PCR)”. This keeps the PCR recitation format the same as for the other listed forms of amplification, and aligns better with the format used in claim 61, which depends on claim 2. Appropriate correction is required.
Claim 24 is objected to because of the following informality: the phrase “DNA-targeting protein” should read “DNA-targeting Cas protein” wherever it appears to better align with the wording used in claim 1, from which this claim depends. Appropriate correction is required.
Claim 56 is objected to because of the following informality: the phrase “further comprising the step of” should read “further comprising a step of.” Appropriate correction is required.
Claim 62 is objected to because of the following informality: the use of “a second droplet” is considered to be a typographical error, as second droplets are already recited in claim 59, from which this claim depends. Therefore, “a second droplet” should read “the second droplet.” Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
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, 2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-57, 61, and 63 are 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.
Claim 1 is rejected because the final newly added wherein clause, “wherein the masking construct prevents CRISPR system activation in the first set of droplets prior to merging with the second set of droplets,” is considered new matter. The instant specification does not describe such a function of the masking construct. Paras. 12 and 74 of the instant specification describe that when a target hybridizes to the guide RNA of a CRISPR system, the CRISPR protein can deactivate the masking construct. Para. 406 in fact describes a masking construct as a “molecule that can be cleaved or otherwise deactivated by an activated CRISPR system effector protein described herein.” This paragraph also generally describes that the function of the masking construct is the blocking or generating of a detectable signal. Neither the claims as filed or the original disclosure describe the masking construct preventing CRISPR system activation.
Claims 2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-57, 61, and 63 are rejected due to their dependence on rejected claim 1.
Claim Rejections - 35 USC § 112(b)
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, 2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-57, and 59-63 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 rejected because of the phrase “wherein the masking construct prevents CRISPR system activation in the first set of droplets prior to merging with the second set of droplets.” Specifically “CRISPR system activation” is not defined by the instant specification, and earlier in the claim, the CRISPR system is stated to comprise the Cas protein, the gRNA, the masking construct, and the first unique optical barcode. It is unknown which particular part of this system must be prevented from being activated by the masking construct, especially given that the system includes the masking construct itself. Additionally, if this phrase is referring to activity of the Cas protein in the CRISPR system to cleave a particular sequence, it is unclear how this cleavage would be initiated without the presence of a sequence to hybridize to the guide RNA in the first droplets. Prior art will be considered to read on this limitation if it discloses a masking construct with a CRISPR-Cas protein and gRNA(s), where the CRISPR-Cas protein does not perform cleavage unless it is in the presence of a target sequence, as this would be analogous to the claimed CRISPR system before it is merged with the contents of the second droplets.
Claims 2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-57, 61, and 63 are rejected due to their dependence on rejected claim 1.
Claim 59 recites “one of the samples” in the final line of the claim. There is insufficient antecedent basis for this limitation in the claim, as a sample, or more than one sample, is not recited earlier in the claim. The samples will be interpreted as containing the target nucleotide sequences, similar to how they are used in instant claim 1. It is recommended that Applicant also specifically state that the second set of droplets contain nucleic acid isolates or extracts from the one or more samples, similar to what is done in claim 1, in order to clearly utilize the samples in the overall multiplex detection system of the claim.
Claims 60 and 62 are rejected based on their dependence on rejection claim 59.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 62 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 62 depends on claim 59, where claim 59 has been amended to require the second optical barcode in the second droplets. As claim 62 also requires that the second droplets contain the second optical barcode, claim 62 fails to further limit the subject matter of the claim upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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.
Claims 59-60 and 62 are rejected under 35 U.S.C. 103 as being unpatentable over Zheng et al. (WO 2017/070056) in view of Blainey et al. (WO 2016/149661), in view of East-Seletsky et al. (Nature, 2016), and in view of Gootenberg et al. (Science, 2017).
Zheng teaches methods and systems for introducing nucleic acid manipulation agents (such as CRISPR systems) into single cells (Abstract and para. 2). This can involve the use of droplets in a microfluidic device, as shown in Figure 1. Cells (character 114) and beads carrying nucleic acid manipulation reagents (character 116) can be carried through microfluidic channels and then pooled together into droplets (paras. 100-101). The cells may be “pre-encapsulated,” and thus can be in droplets before meeting with the reagents (paras. 59, 61, and 100). Once inside the droplets, cells can be lysed before further analysis occurs, thus extracting the nucleic acids for use, particularly to measure target expression (para. 66). When CRISPR systems are used, guide RNAs (gRNAs) can be found on the beads in droplets (para. 82). Zheng has multiple instances of teaching droplets in emulsions, and teaches such emulsion droplets for both the nucleic acids and the CRISPR reagents (e.g. paras. 29, 39, 53, 58-59). Multiple different cells types can be used (para. 57), and multiple organisms can be used (para. 117), meaning multiple samples can be used. Zheng also teaches that anywhere from 2-10,000 different guide RNAs may be used in their invention (para. 83). Each gRNA can have a barcode label that is fluorescent (para. 83), such as a fluorophore that may be detected by optical means, thus making it an optical label (para. 88). In CRISPR systems, Cas9, C2c2 (i.e. Cas13), or Cpf1 (i.e. Cas12) may also be used (paras. 10, 78, 103, and 128-129). Zheng teaches that detection of the detectable barcodes can occur, and that multiple labels can be detected via a detectable fluorescent signal (paras. 87-88).
However, Zheng does not teach that the CRISPR system may have a masking construct, that the target cells may have optical barcodes, that the microfluidic device can deposit the pooled droplets into microwells, or that multiple detection reactions may occur simultaneously. The reference also does not teach measuring sensitivity on an attomolar scale.
Blainey teaches use of microfluidic devices for on-chip screening of combinatorial libraries and methods of use thereof (Abstract). The microfluidic device has an input for receiving multiple droplets, and an array of microwells for randomly receiving two or more droplets from the microfluidic device (para. 3). The droplets may flow into the microwells via buoyancy from the microfluidic device situated underneath the microwells (para. 5). The droplets in each microwell are then merged (para. 5).In Figure 2 of Blainey, two droplets are shown in a partition as separate entities, showing that they may exist as such without immediately merging when entering a partition. Figures 4 and 11 also show imaging of the microwells both pre- and post-merging. Para. 52 notes that merging can be initiated by a particular device or reaction, and so merging need not occur upon capture of the droplets in the microwells. The droplets in the microfluidic device may be attached to an optical barcode that identifies the molecular species of interest, as well as a reporter agent for detection (para. 5). The reporter may be an optically labeled nucleic acid used to detect an agent in the merged droplet that results from the combination of two molecular species, or from the generation of a particular product, thus also acting as an optical barcode (para. 49). After droplet merging in the microwell, said microwell can be optically scanned to read the provided barcodes and measure the reporter of each merged droplet simultaneously (para. 53). The number of microwells imaged can be 15 times the number of droplets generated (para. 51). Since each microwell holds only two droplets, every microwell can be imaged at one time.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to combine the teachings of Zheng and Blainey. Specifically, the ordinary artisan would be capable of using the general droplet/merging methodology of Zheng (where CRISPR systems for multiple types of cells/organisms, along with an optical barcode, can be combined with their targets in droplets) with the microwell array droplet merging and simultaneous imaging/detection of Blainey (where all droplets may have optical barcodes). Zheng’s methods mainly concern allowing nucleic acid manipulation reagents (i.e. CRISPR systems) into partitions for particular targets, and notes that their methods can be used in screening processes (Abstract and para. 3). Blainey teaches such screening processes, and notes that their method is more robust and easily scalable compared to similar methods (Abstract and para. 2). By combining these methods, it would allow the ordinary artisan to image the target nucleic acids, which would aid in determining if particular mutants are present in a sample, or if desired genetic manipulations have occurred. Blainey also specifically teaches that targets may have optical barcodes, and incorporating these and detecting them in the method of Zheng would allow for increased accuracy in the detection of droplet contents, and would give the ordinary artisan the ability to note if a target nucleic acid, a CRISPR system, or both are present in a merged droplet (instant claim 62). By providing droplets with targets, droplets with CRISPR systems, and merging droplets, at least some of the merged droplets will contain both components. Regarding the language of instant claim 59, those droplets which do contain both components can be considered the “pool of merged droplets.” MPEP 2143 I (A) states, “The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art.” Droplet and CRISPR systems, optical barcodes, and microwell imaging are well-known in the art, as evidenced by Zheng and Blainey, and this combination would simply provide droplets with specific contents to merge and image as taught by Blainey, without changing the overall methodology. Therefore, these results would be predictable.
However, Blainey does not teach that the CRISPR system may include a masking construct, nor does the reference teach measuring sensitivity on an attomolar scale.
East-Seletsky teaches the RNase activity of C2C2 in a multiplex capacity (Abstract). As part of the testing of C2C2, East-Seletsky teaches the use of reporter RNA added to the CRISPR system. This reporter is fluorophore-quencher labeled, and once the target RNA is bound to the guide RNA, the reporter is cleaved, separating the fluorophore and quencher and allowing for increased fluorescence detection (Figure 4A and page 272, column 1, para. 1). The reporter thus suppresses a positive fluorophore signal until a target RNA sequence is present.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use reporter RNAs as taught by East-Seletsky in the method of Zheng in view of Blainey. These reporters would allow for significantly increased signal only when the CRISPR system is in the merged droplet with the target nucleic acid sequence, as target hybridization is required to produce a strong signal. Thus, the reporters can aid in determining how many desired merged droplets are present in an imaged microwell. East-Seletsky also teaches the use of said reporter in a CRISPR system, showing that the addition of this reporter would not interfere with other aspects of the CRISPR system and overall method, as it could be designed to avoid hybridization to target sequences that hybridize to other system components, such as the gRNAs. The results of this addition would therefore be predictable with a reasonable expectation of success.
However, East-Seletsky does not teach measuring sensitivity on an attomolar scale.
Gootenberg teaches nucleic acid detection with CRISPR systems, and analyses the sensitivity of several amplification methods in association with this detection. This primarily relied on SHERLOCK methods, which use amplification and CRISPR systems to detect targets (Figure 1). SHERLOCK with recombinase polymerase amplification (RPA) in general is taught to have attomolar sensitivity (Figure 1C and page 2, column 1, para. 2). Gootenberg amplified SHERLOCK products via PCR, and found that target molecules could be detected with attomolar sensitivity as well (Figure 2C and D and page 2, column 1, para. 3). This reference concludes that combining CRISPR with amplification can be useful for detecting nucleic acid contamination, disease identification, and genotyping, and is a relatively inexpensive and rapid method with high sensitivity and specificity (page 4, final para.).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to combine the methods of Zheng, in view of Blainey, and in view of East-Seletsky with those of Gootenberg to add amplification of target sequences to the method and arrive at the method of claims 59 and 62. Blainey already teaches adding amplification reagents to merged droplets, and the teachings of Gootenberg would prompt the ordinary artisan to amplify target sequences, not just optical barcode sequences. Gootenberg teaches that PCR and RPA can be used with CRISPR methods, both with attomolar sensitivity, and states the many benefits and uses of combining these methodologies. The reference also teaches methods of primer design with publicly available software (Supplementary Materials, page 4, “Recombinase Polymerase Amplification”) and commercially available primers (Supplementary Materials, page 5, “Digital droplet PCR quantification”), thus showing that the ordinary artisan would be capable of designing or obtaining primers for known target sequences. MPEP 2143 I (A) states, “The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art.” As primer design and amplification are both known methods that have predictable results, this combination would also be predictable, and would simply result in the amplification of both barcode and target sequences.
Thus, claims 59 and 62 are prima facie obvious over Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg.
Regarding claim 60, Zheng teaches that the microfluidic device, reagents, and other necessary components for performing their invention may be provided in a kit (para. 121). Therefore, it would be prima facie obvious to include the components of the system of Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg in such a kit as well, in order to allow for commercial sale of the invention and ease of shipping.
Claims 1, 2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-55, 57, 61, and 63 are rejected under 35 U.S.C. 103 as being unpatentable over Zheng et al. (WO 2017/070056), in view of Blainey et al. (WO 2016/149661), in view of East-Seletsky et al. (Nature, 2016), and in view of Gootenberg et al. (Science, 2017).
Zheng teaches methods and systems for introducing nucleic acid manipulation agents (such as CRISPR systems) into single cells (Abstract and para. 2). This can involve the use of droplets in a microfluidic device, as shown in Figure 1. Cells (character 114) and beads carrying nucleic acid manipulation reagents (character 116) can be carried through microfluidic channels and then pooled together into droplets (paras. 100-101). The cells may be “pre-encapsulated,” and thus can be in droplets before meeting with the reagents (paras. 59, 61, and 100). Once inside the droplets, cells can be lysed before further analysis occurs, thus extracting the nucleic acids for use, particularly to measure target expression (para. 66). When CRISPR systems are used, guide RNAs (gRNAs) can be found on the beads in droplets (para. 82). Zheng has multiple instances of teaching droplets in emulsions, and teaches such emulsion droplets for both the target nucleic acid and the CRISPR reagents (e.g. paras. 29, 39, 53, 58-59). The gRNAs can be specific to a disease loss of function gene or used to inactivate a disease gene (para. 83; instant claim 12). The target cells may be bacterial, yeast, or human cells (instant claim 5). Multiple different cells types can be used (para. 57), and multiple organisms can be used (para. 117), meaning multiple samples can be used. Zheng also teaches that anywhere from 2-10,000 different guide RNAs may be used in their invention (para. 83). Each gRNA can have a barcode label that is fluorescent (para. 83), such as a fluorophore that may be detected by optical means, thus making it an optical label (para. 88). The gRNAs can introduce alterations in target sequences (paras. 51-52), and thus can have mismatches to the target sequence (instant claim 8). In CRISPR systems, Cas9, C2c2 (i.e. Cas13), or Cpf1 (i.e. Cas12) may also be used (paras. 10, 78, 103, and 128-129; instant claim 24). Zheng teaches that detection of the detectable barcodes can occur, and that multiple labels can be detected via a detectable fluorescent signal (paras. 87-88; instant claim 55).
However, Zheng does not teach that the CRISPR system may have a masking construct, that the target cells may have optical barcodes, that the microfluidic device can deposit the pooled droplets into microwells, or that multiple detection reactions may occur simultaneously. The reference also does not teach detecting targets with attomolar sensitivity.
Blainey teaches use of microfluidic devices for on-chip screening of combinatorial libraries and methods of use thereof (Abstract). The microfluidic device has an input for receiving multiple droplets, and an array of microwells for randomly receiving two or more droplets from the microfluidic device (para. 3). The droplets may flow into the microwells via buoyancy from the microfluidic device situated underneath the microwells (para. 5). The droplets in each microwell are then merged (para. 5). In Figure 2 of Blainey, two droplets are shown in a partition as separate entities, showing that they may exist as such without immediately merging when entering a partition. Figures 4 and 11 also show imaging of the microwells both pre- and post-merging. Para. 52 notes that merging can be initiated by a particular device or reaction, and so merging need not occur upon capture of the droplets in the microwells. The droplets in the microfluidic device may be attached to an optical barcode that identifies the molecular species of interest, as well as a reporter agent for detection (para. 5). The reporter may be an optically labeled nucleic acid used to detect an agent in the merged droplet that results from the combination of two molecular species, or from the generation of a particular product, thus also acting as an optical barcode (para. 49). Imaging can occur at multiple time points to track changes in a reporter over time (para. 54). After droplet merging in the microwell, said microwell can be optically scanned to read the provided barcodes and measure the reporter of each merged droplet simultaneously (para. 53). The number of microwells imaged can be 15 times the number of droplets generated (para. 51). Since each microwell holds only two droplets, every microwell can be imaged at one time.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to combine the teachings of Zheng and Blainey. Specifically, the ordinary artisan would be capable of using the general droplet/merging methodology of Zheng (where CRISPR systems for multiple types of cells/organisms, along with an optical barcode, can be combined with their targets in droplets) with the microwell array droplet merging and simultaneous imaging/detection of Blainey (where all droplets may have optical barcodes). Zheng’s methods mainly concern allowing nucleic acid manipulation reagents (i.e. CRISPR systems) into partitions for particular targets, and notes that these methods can be used in screening processes (Abstract and para. 3). Blainey teaches such screening processes, and notes that their method is more robust and easily scalable compared to similar methods (Abstract and para. 2). By combining these methods, it would allow the ordinary artisan to image the sample cells, which would aid in determining if particular mutants are present in a sample, or if desired genetic manipulations have occurred. Blainey also specifically teaches that targets may have optical barcodes, and incorporating these and detecting them in the method of Zheng would allow for increased accuracy in the detection of droplet contents, and would give the ordinary artisan the ability to note if a target nucleic acid, a CRISPR system, or both are present in a merged droplet. By providing droplets with targets, droplets with CRISPR systems, and merging droplets, and least some of the merged droplets will contain both components, as noted in instant claim 1. MPEP 2143 I (A) states, “The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art.” Droplet and CRISPR systems, optical barcodes, and microwell imaging are well-known in the art, as evidenced by Zheng and Blainey, and this combination would simply provide droplets with specific contents to merge and image as taught by Blainey, without changing the overall methodology. Therefore, these results would be predictable.
Additionally, though Blainey does not explicitly state in their methods that droplets may be imaged in the microwells prior to merging (thus detecting the optical barcodes when in the individual droplets), the reference shows imaging of the droplets in the microwells prior to merging, and in Figure 11 in particular, different droplets appear to be imaged with different fluorescent values. The ordinary artisan would recognize that in Zheng in view of Blainey, it would generally be helpful to image the droplets both pre- and post-merging, in order to: 1) determine the accuracy/effectiveness of the merging methodology used; 2) ensure that optical barcodes are individually detectable and distinguishable before merging, so that both CRISPR systems and targets will be easily visible in the merged droplets; and 3) provide an estimate of the abundance of particular targets in samples relative to the CRISPR system for those targets present in the microwells, thus allowing the ordinary to fine tune the multiplexing of the microwells to a level that would be most effective for detection of all desired targets. Blainey also teaches that the optical barcodes in the microwells can be detected with imaging microscopy (Abstract). This reference also shows examples of fluorescent microscopy (Figure 9 and paras. 16, 46, and para. 66 “Imaging and analysis”). Though this fluorescent microscopy is generally for the post-merging imaging, it would also be prima facie obvious to use the same imaging methods both pre- and post-merging to prevent the need for additional imaging materials or equipment. There would be a reasonable expectation of success as Blainey already shows effective use of fluorescent imaging (e.g. Figure 7 and 9), and so this would simply involve performing the known imaging method of Blainey at an additional time point (instant claim 48).
However, Blainey does not teach that the CRISPR system may include a masking construct, nor does this reference discuss detecting target sequences with attomolar sensitivity. It is noted that Blainey teaches that primers amplification reagents may be delivered to merged droplets in order to amplify the optical barcode (paras. 54 and 57), but the reference does not specifically teach amplifying target sequences.
East-Seletsky teaches the RNase activity of C2C2 in a multiplex capacity (Abstract). As part of the testing of C2C2, East-Seletsky teaches the use of reporter RNA added to the CRISPR system. This reporter is fluorophore-quencher labeled, and once the target RNA is bound to the guide RNA, the reporter is cleaved, separating the fluorophore and quencher and allowing for increased fluorescence detection (Figure 4A and page 272, column 1, para. 1). The reporter thus suppresses a positive fluorophore signal until a target RNA sequence is present.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use reporter RNAs as taught by East-Seletsky in the method of Zheng in view of Blainey. These reporters would allow for significantly increased signal only when the CRISPR system is in the merged droplet with the target nucleic acid sequence, as target hybridization is required to produce a strong signal. Thus, the reporters can aid in determining how many desired merged droplets are present in an imaged microwell. East-Seletsky also teaches the use of said reporter in a CRISPR system, showing that the addition of this reporter would not interfere with other aspects of the CRISPR system and overall method, as it could be designed to avoid hybridization to target sequences that hybridize to other system components, such as the gRNAs. The results of this addition would therefore be predictable with a reasonable expectation of success (instant claims 29, 40, and 47). Additionally, it is noted that this masking construct meets the newly added limitation of claim 1 in accordance with the interpretation provided in the 35 USC 112(b) Rejection above, as the Cas protein does not cleave the masking construct until a target nucleic acid is hybridized to the gRNA, and in Zheng, in view of Blainey, and in view of East-Seletsky, this would not occur until the first and second droplets are merged, as the second droplets would contain the potential target sequences.
However, East-Seletsky does not teach measuring sensitivity on an attomolar scale.
Gootenberg teaches nucleic acid detection with CRISPR systems, and analyses the sensitivity of several amplification methods in association with this detection. This primarily relied on SHERLOCK methods, which use amplification and CRISPR systems to detect targets (Figure 1). SHERLOCK with recombinase polymerase amplification (RPA) in general is taught to have attomolar sensitivity (Figure 1C and page 2, column 1, para. 2). Gootenberg amplified SHERLOCK products via PCR, and found that target molecules could be detected with attomolar sensitivity as well (Figure 2C and D and page 2, column 1, para. 3). This reference concludes that combining CRISPR with amplification can be useful for detecting nucleic acid contamination, disease identification, and genotyping, and is a relatively inexpensive and rapid method with high sensitivity and specificity (page 4, final para.).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to combine the methods of Zheng, in view of Blainey, and in view of East-Seletsky with those of Gootenberg to add amplification of target sequences to the method and arrive at the method of instant claims 1, 2, 61, and 63. Blainey already teaches adding amplification reagents to merged droplets, and the teachings of Gootenberg would prompt the ordinary artisan to amplify target sequences, not just optical barcode sequences. Gootenberg teaches that PCR and RPA can be used with CRISPR methods, both with attomolar sensitivity, and states the many benefits and uses of combining these methodologies. The reference also teaches methods of primer design with publicly available software (Supplementary Materials, page 4, “Recombinase Polymerase Amplification”) and commercially available primers (Supplementary Materials, page 5, “Digital droplet PCR quantification”), thus showing that the ordinary artisan would be capable of designing or obtaining primers for known target sequences. MPEP 2143 I (A) states, “The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art.” As primer design and amplification are both known methods that have predictable results, this combination would also be predictable, and would simply result in the amplification of both barcode and target sequences.
Thus, claims 1, 2, 5, 8, 12, 24, 29, 40, 47-48, 55, 61, and 63 are prima facie obvious over Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg.
Regarding claims 10 and 15, neither Zheng, nor Blainey, nor East-Seletsky teach that the guide RNAs are specifically designed to detect SNPs.
Gootenberg teaches discriminating between Zika and Dengue viral strains that contain SNPs and sequences of the crRNAs (part of gRNAs) that are used for this discrimination (Figure 3). Incorporating SNP nucleotides into the crRNA improves detection of particular strains (Figures 3B and D). Figure 4 also shows that crRNAs can also be developed to detect human SNPs.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Gootenberg to design at least some of the gRNAs in the method of Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg to specifically detect viral and human targets with an SNP. This targeting could alert practitioners to the presence of particular genetic mutations in the analyzed samples, as the first optical barcode and fluorophore associated with the masking construct would give off fluorescence when in the presence of the target mutant. As Zheng teaches that samples can come from humans (e.g. para. 57), this method could be used to diagnose genetic mutations in humans and demonstrate if genetic alteration is possible, as well as to determine what particular strain of a virus an individual may be infected with, both of which would be of interest to patients and clinicians. Gootenberg teaches that gRNAs may be designed based on known sequences (e.g. Figure 3), as well as provides evidence that SNPs can be targeted, thereby providing a reasonable expectation of success.
Thus, claims 10 and 15 are prima facie obvious over Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg.
Regarding claim 14, Zheng teaches analyzing bacterial cells (para. 57). Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Gootenberg in the method of Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg to arrive at the invention of claim 14. Specifically, Gootenberg, as noted above, demonstrates that crRNAs, which are part of gRNAs, can be designed to detect particular strains of infection. By doing this with bacterial cells, such as those taught by Zheng, one could determine if antibiotically resistant infectious bacteria are present in a sample, or if particular bacterial strains are present that respond differently to different treatments. If the sample is from a patient, this information would be valuable in developing treatment plans. Gootenberg also teaches that their method is effective, is able to be cheaply redesigned, and has high sensitivity and specificity. Gootenberg teaches that gRNAs may be designed based on known sequences (e.g. Figure 3), and gRNA design is well-known, as evidenced by Zheng and Gootenberg, providing a reasonable expectation of success.
Therefore, claim 14 is prima facie obvious over Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg.
Regarding claim 17, Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg teaches the method of claim 15, as described above. Zheng also teaches that anywhere from 2-10,000 different guide RNAs may be used in their invention, as noted above in the rejection of claim 1 (para. 83).
Regarding claim 50, Blainey teaches that optical barcodes “can be defined from a set of objects, such as beads, that vary in size, shape, color, or a combination thereof,” (para. 46). These beads consisting of different parameters would increase the number of distinct barcodes that could be used. Thus, it would be prima facie obvious for the ordinary artisan to use this barcoding methodology in Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg in order to be able to distinctly label and detect more target sequences at one time compared to using single dyes and single type beads for each target. As beads can be commercially purchased and procedures exist for loading the beads with different dyes (e.g. Blainey para. 66 under “Bead production for optical barcoding”), the ordinary artisan would have a reasonable expectation of success, as they would simply need to purchase the needed dyes and beads of different sizes and shapes.
Regarding claims 53-54, Blainey teaches that optical barcodes can comprise a sub-set of fluorophores, where distinct colors may be generated by varying the ratio of multiple dyes (para. 46 and Figures 5 and 8). Blainey teaches that utilizing ratios of dyes can provide more distinct colors for barcodes than if the dyes were used alone. Thus, it would be prima facie obvious for the ordinary artisan to use this method in Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg in order to be able to distinctly label and detect more target sequences at one time compared to using single dyes for each target. Figure 8 shows experimental use of this method, providing a reasonable expectation of success.
Regarding claim 57, Blainey teaches that the number of microwells provided can be up to 1,000,000 (para. 36). The ordinary artisan would recognize the value of including multiple wells in the array of Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg, as this would allow for more opportunity for the desired droplet pairs to merge with one another. Additionally, as more samples/target nucleic acids are used or desired, additional microwells would ensure that each target, if present in the sample, is more likely to be detected by the appropriate CRISPR system. Additional microwells also allow for replicate testing of particular targets. Thus, as Blainey teaches the use of up to 1,00,000 microwells, the ordinary artisan would be motivated to include this many microwells in Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg.
Claim 56 is rejected under 35 U.S.C. 103 as being unpatentable over Zheng et al. (WO 2017/070056), in view of Blainey et al. (WO 2016/149661), in view of East-Seletsky et al. (Nature, 2016), in view of Gootenberg et al. (Science, 2017), and further in view of Jabado et al. (Nucleic Acids Research, 2008).
Regarding claim 56, Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg teach the method of claim 1, as described above. However, none of these references teach a set cover solving process.
Jabado teaches methods of probe design using the set cover algorithm (Abstract and page 3, column 1, para. 2). This algorithm allows for the design of the minimum set of probes required to detect a set of target sequences. Jabado explains an overview of the analysis and the parameters chosen for the probes (page 3, column 1, para. 2). Jabado also teaches methods to verify the set coverage probes (Figure 3) and teaches that while the use of this algorithm may increase computational resources needed to generate probe sets, the set coverage probes have more coverage of target sequences compared to tiling based methods (pages 6-7, “Motif-based probe design provides higher coverage than virus genome tiling”). Jabado also notes that “The method of probe design and set cover minimization is flexible and agnostic of platform; application to bead, solution, or surface-based hybridization technology should be straightforward,” (page 9, column 1, para. 1).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the set cover algorithm taught by Jabado in the method of Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg. This would allow for design of the minimum amount of distinct gRNAs needed to successfully hybridize to the distinct targets in the method of Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg. Jabado teaches that set cover methods allow for better overall coverage, and so allow for better probe/target (or in this case, gRNA/target) hybridization. Since the set cover algorithm is entirely computational, it can be done before the method of Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg, and would not interfere with any of the non-computational method steps. By using this algorithm to design the gRNAs and minimize the number needed, it would cut down on experimental resources and reagents needed to perform the method of Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg, and would thus be of interest for the ordinary artisan to employ. As the set cover method is a known algorithm, there would be a reasonable expectation of success.
Thus, claim 56 is prima facie obvious over Zheng, in view of Blainey, in view of East-Seletsky, in view of Gootenberg, and further in view of Jabado.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681