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 .
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claim 64 has been added. Claims 30-34, 41, and 44-45 remain withdrawn.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 59-64 are considered on the merits herein.
Response to Applicant’s Amendments
Claim Objections
Claims 1-2, 24, 56, and 62 were objected to for various informalities. In light of Applicant’s amendments to the claims submitted 5/26/2026, these objections have been withdrawn. However, see new grounds of objection below.
35 USC 112(a) Rejections
Claims 1-2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-57, 61, and 63 were rejected for a new matter issue associated with claim 1. In light of Applicant’s amendments to the claims submitted 5/26/2026, this rejection has been withdrawn.
35 USC 112(b) Rejections
Claims 1-2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-57, and 59-63 were rejected due to various indefiniteness issues. In light of Applicant’s amendments to the claims submitted 5/26/2026, this rejection has been withdrawn.
35 USC 112(d) Rejections
Claim 62 was rejected for failing to further limit the subject matter of the claim upon which it depended. In light of Applicant’s amendments to the claims submitted 5/26/2026, this rejection has been withdrawn.
35 USC 103 Rejections
Claims 59-60 and 62 were 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).
Applicant has not provided any arguments against these specific rejections, and the amendment to claim 59 does not obviate the current prior art rejections. Therefore, the rejections for claims 59-60 have been maintained. Claim 62 has been amended to require new grounds of rejection, which are provided below.
Claims 1, 2, 5, 8, 10, 12, 14-15, 17, 24, 29, 40, 47-48, 50, 53-55, 57, 61, and 63 were 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).
Applicant’s arguments and amendments have been thoroughly reviewed and considered. These rejections have been maintained. See “Response to Applicant’s Arguments” below. See also new grounds of rejection below for newly added claim 64.
Claim 56 was 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).
Applicant’s arguments and amendments have been thoroughly reviewed and considered. These rejections have been maintained. See “Response to Applicant’s Arguments” below.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections, Applicant argues that Blainey does not teach that the optical barcodes in the first and second droplets are detected pre-merging, and further states that the reference only teaches such detection after droplets are merged. Thus, the use of this reference in combination with Zheng does not meet the requirements of determining obviousness under MPEP 2143 I (A). Applicant recites portions of Blainey that explicitly state the detection of optical barcodes post-merging. Applicant also argues that Figure 11 of Blainey does not image barcodes pre-merging in a manner that would read on the instant claims (i.e. “a deliberate execution of a fluorescence imaging protocol to detect and record optical barcodes,” Remarks, page 13, para. 2). Thus, Applicant argues that the Examiner mischaracterizes the reference (Remarks, pages 11-14).
In considering instant claim 1, the amendment to the detecting step now explicitly requires detecting the first and second unique optical barcodes in their respective first and second droplets. However, the “detecting” step is not made specific. Para. 16 of the instant specification notes that detection of such a barcode can occur “by using light microscopy, fluorescence microscopy, Raman spectroscopy, or a combination thereof,” but this is not a limiting recitation. The “detecting” itself also has no specific definition in the instant specification. Thus, this detection may be any method by which the presence of an optical barcode in a droplet may be confirmed – no particular readout of the barcode is required.
In reciting teachings of Blainey in the Non-Final Rejection that could be used to read on the pre-merging detection of the instant claims, Figures 4 and 11 were recited. Figure 4 clearly shows droplets pre- and post-merging (though this figure is not focused on potential barcodes within the droplets), and Figure 11 is stated in para. 18 to be “a set of images showing an example microwell designed to contain 6 drops, pre-merge (top) and post -merge (bottom).” The figure itself shows a set of droplets with distinct optical differences between them, as evidenced by the different shades of the droplets. Para. 46 (cited in the Non-Final Rejection) of Blainey also notes that the optically detectable barcodes can be visualized with light alone.
Blainey’s clear teachings regarding imaging the droplets and optical barcodes post-merging do not indicate that the teachings of the reference may not be used for other detection means. The reference does not teach away from pre-merge detection, or provide any reason why such detection should be discouraged or not used. 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.” Additionally, MPEP 2144 I states, “The rationale to modify or combine the prior art does not have to be expressly stated in the prior art; the rationale may be expressly or impliedly contained in the prior art or it may be reasoned from knowledge generally available to one of ordinary skill in the art, established scientific principles, or legal precedent established by prior case law. In re Fine, 837 F.2d1071, 5 USPQ2d 1596 (Fed. Cir. 1988),”
While Applicant states that the obviousness rejection of Zheng in view of Blainey is based on the guidance of MPEP 2143 I (A), this is only a portion of the rejection (see para. 67 of the Non-Final Rejection). Para. 68 of the rejection specifically addresses the pre-merging detection of the instant claims. This addresses the teachings the ordinary artisan would glean from Blainey, and provides additional motivation for pre-merging imaging that the ordinary artisan would glean through ordinary creativity, skill, and knowledge in the art. Specifically, para. 68 states, “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.” A reasonable expectation of success was also provided. Applicant does not specifically address these points of the rejection.
Thus, it is the Examiner’s position that, given the teachings of Blainey, along with the ordinary artisan’s capabilities, Zheng in view of Blainey would arrive at the claimed pre-merging detection. The fact that this combination and obviousness determination/rationale does not rely on an explicit teaching in the reference does not obviate the rejection.
Thus, Applicant’s arguments are not considered persuasive to overcome the rejections associated with claim 1 and its dependent claims. Thus, these rejections have been maintained for claim 1 and all of its dependent claims, with the exception of newly added claim 64, for which new grounds of rejection are provided below. It is noted that the East-Seletsky reference already addresses the “suppresses a detectable signal” language of newly amended claim 1 (see paras. 56-57 of the Non-Final Rejection), and Applicant has not provided any arguments against this reference.
It is noted that claim 59 does not require the pre-merging detection of claim 1, and Applicant has not provided any arguments that appear to specifically address this claim. Therefore, the rejections of claim 59 and all of its dependent claims have been maintained, with the exception of amended claim 62, for which new grounds of rejection are provided below.
Claim Objections
Claim 62 is objected to because of the following informality: in lines 1-2, “wherein the the second droplet” should read “wherein the second optical barcode of the second droplet”. Appropriate correction is required.
Claim 64 is objected to because of the following informality: in line 1, “the RNA-targeting protein” should read “the RNA-targeting Cas protein” for clarity. Appropriate correction is required.
Claim Interpretation
Regarding the use of the phrase “separate entities,” in the wherein clause associated with the first detecting step 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 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. 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 claim 59. 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, claim 59 is 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.
Regarding claim 62, 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.
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.
Claim 64 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), and in view of Gootenberg et al. (Science, 2017), as evidenced by Cox et al. (Science, 2017; cited in a previous Office Action).
Regarding claim 64, Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg teach the method of claim 1, as described above. Zheng also teaches that Cas13 (C2c2) may be used in their method (para. 78; see this teaching above in the rejection of claim 1). As evidenced by Cox, this protein has two HEPN domains (page 2, para. 2).
Therefore, claim 64 is prima facie obvious over Zheng, in view of Blainey, in view of East-Seletsky, and in view of Gootenberg, as evidenced by Cox, for the same reasons described above for claim 1.
Conclusion
No claims are currently allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/F.F.G./Examiner, Art Unit 1681
/GARY BENZION/Supervisory Patent Examiner, Art Unit 1681
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