DETAILED ACTION
The amendment filed on 03/04/2026 has been entered.
Claims 1, 32, 57, 80, and 82 were amended in the claim set filed on 03/04/2026.
Applicant’s election without traverse of Group I, claims 1-2, 7, 13, 17, 25, 27, 30, 32, 34, 46, 57, 61-62, 77-78, 80, 82, 85-87, 89, 97 and 99-101, drawn to methods of pooled optical screening of genetically barcoded cells comprising genetic perturbations and simultaneous transcriptional measurements in the reply filed on 09/16/2025 is acknowledged.
Claims 117 and 145 are withdrawn drawn to a nonelected Group II.
Claims 147-148 were added in the claim set filed on 03/04/2026. No new matter was added.
Claims 1-2,7,13,17,25,27,30,32,34,46,57,61-62,77-78,80,82,86-87,89,97,99-101 and 147-148 in the claim set filed on 03/04/2026 are currently under examination.
Response to the Arguments
Objections to the claim 32 in the previously mailed non-final has been withdrawn in light of applicants claim amendments.
Applicant’s arguments regarding previous rejection(s) of claim(s) 85 under 35 U.S.C. 112 have been fully considered and are moot, in light of cancellation of claim.
Applicant’s arguments regarding previous rejection(s) of claim 101 under 35 U.S.C. 112 have been fully considered but are not persuasive. The 35 U.S.C. 112 rejection for claim 101 documented in the previously mailed non-final has been maintained and revised in light of applicants claim amendments and arguments on Pg. 11-12. Furthermore, as necessitated by amendment to claims 1 and 80, and newly added claim 148, new grounds of rejection are made as documented below in the 35 U.S.C. 112 (b) rejection in this office action on Pg. 4-5.
Applicant’s arguments regarding previous rejection(s) of claim(s) 80 and 82 under 35 U.S.C. 112 have been fully considered and are persuasive. The 35 U.S.C. 112 rejections documented in the previously mailed non-final have been withdrawn, in light of applicants claim amendments and arguments on Pg. 12.
As necessitated by amendment, the 35 U.S.C. 102 rejections of claim(s) 1-2, 7, 13, 17, 25, 27, 61-62, 77-78, 80, 82, 97 and 99-101 documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 11-12. However, upon further consideration and search new grounds of rejection under 35 U.S.C. 103 have been made, as documented below in this office action on Pg. 6-19.
As necessitated by amendment, the 35 U.S.C. 103 rejections of claim(s) 1, 30, 32, 34, 46, 57, 80, 86-87and89 documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 12-16. However, upon further consideration and search new grounds of rejection under 35 U.S.C. 103 have been made, as documented below in this office action on Pg. 19-31.
As necessitated by amendment, new grounds of 35 U.S.C. 103 rejections of newly added claim(s) 147-148 are made, as documented below in the 35 U.S.C. 103 rejection in this office action on Pg. 19.
The rejections for claims 1-2,7,13,17,25,27,30,32,34,46,57,61-62,77-78,80,82,86-87,89,97,99-101 and 147-148 are documented below in this Final Office Action are necessitated by claim amendments filed on 03/04/2026.
Priority
This application claims priority benefit to U.S. Provisional Application No. 63/313,189, filed on February 23, 2022, the contents of which are hereby incorporated by reference in their entirety for all purposes. The priority date of claim set filed on June 20, 2023, is determined to be Feb. 23, 2022.
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.
Claim 1-2,7,13,17,25,27,30,32,34,46,57,61-62,77-78,80,82,86-87,89,97,99-101 and 147-148 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is indefinite over the limitation “(a)(ii) preserving the barcode sequence of the at least one cell before completing (b)” and “performing at least one round of fluorescence in situ hybridization (FISH)”. It is unclear whether the preservation occurs before step (b), during step (b), or both before and during step (b). If during step (b), it is unclear if the preservation occurs after one round of step (b), after each round of step (b) or after multiple rounds of step (b) or whether the preservation occurs during a single round of step (b), alternatively during each round of step (b), and not after a round of step (b). Claims 2,7,13,17,25,27,30,32,34,46,57,61-62,77-78,80,82,86-87,89,97,99-101 and 147-148 depend on claim 1.
Claim 80 is indefinite over the limitation “The method of claim 32, wherein (a)(ii) is performed after step (iv) and before step (v)”. It is unclear as written if (a)(ii) is referring to claim 32 (ii) or claim 1 (a)(ii). Step (ii) of claim 32 is drawn to the limitation of “contacting the plurality of mRNA transcripts comprising the at least one mRNA transcript with a plurality of 3' probes, wherein each 3' probe comprises a second target hybridization sequence complementary to a second portion of the mRNA transcript of the plurality of mRNA transcripts, wherein each 3' probe is capable of specifically hybridizing with a 5' loop probe, wherein hybridization of a 3' probe with a corresponding 5' loop probe forms a loop in the 5' loop probe” while Step (a)(ii) is drawn to the limitation “preserving the barcode sequence of the at least one cell before completing (b)”.
Claim 101 is indefinite over the limitation “receiving a first image depicting the plurality of cells, wherein the first image indicates each cell of the plurality of cells by a boundary and associates each cell of the plurality of cells with a corresponding cell identifier; receiving a second image depicting locations of a plurality of barcode sequences, wherein the plurality of barcode sequences are associated with the plurality of cells after POSH is performed”, “receiving a FISH image of the plurality of cells after the at least one round of FISH is performed on the plurality of cells”, “based on the alignment between the first image and the FISH image, resizing the FISH image” and “associating each cell of the plurality of cells with a portion of the resized FISH image, the corresponding barcode sequence, and the corresponding cell identifier; and analyzing known phenotypes or identifying new phenotypes of the plurality of cells”. It is unclear as written how this “FISH image” is the same as or different than the first image or second image, and exactly what the FISH image correlates to.
Claim 148 recites the limitation " the reverse transcribed barcode sequence of the at least one cell" in ln 2. The limitations of Claim 1, in which claim 148 depends on does not comprise a limitation requiring a reverse transcribed barcode. There is insufficient antecedent basis for this limitation in the claim.
Response to Arguments
Applicant's arguments filed 03/04/2026, do not apply to the new grounds of rejection with regards to claims 1-2,7,13,17,25,27,30,32,34,46,57,61-62,77-78,80,82,86-87,89,97,99-101 and 147-148 under 35 USC 112(b), as necessitated by amendment. Applicant's arguments filed 03/04/2026, with regard to previously rejected claim 101, have been fully considered but are not persuasive. To clarify some instances argued in the response filed 03/04/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “Applicant believes that the claim is clear. Features and/or uses of the various images are clearly indicated in the claim. ”(Pg. 12).
Response: In response to the argument stated above, it is unclear as written how the “FISH image” is the same as or different than the first image or second image, and exactly what the FISH image correlates to.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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 1-2, 7, 13, 17, 25, 27, 61-62, 77-78, 82, 97, 99-101 and 147-148 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. “Wang”; (2019). Proceedings of the National Academy of Sciences of the United States of America (PNAS), 116(22), 10842–10851., Published online May 13, 2019) in view of Feldman et al. “Feldman”; (2022). Pooled genetic perturbation screens with image-based phenotypes. Nature protocols, 17(2), 476–512., Published online Jan. 12, 2022).
Wang discloses “Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are co-delivered to cells with corresponding barcodes placed at the 3′ untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy.” (Abstract).
Regarding claim 1, Wang teaches a method wherein “pooled-library CRISPR screening in mammalian cells. This approach allows both high-content phenotype imaging of multiple molecular targets in individual cells and high-accuracy identification of the genotype of each cell, the latter achieved by associating each sgRNA with unique barcodes and reading out the barcodes using multiplexed error-robust fluorescence in situ hybridization (MERFISH)” (Pg. 10842, Col. 2, Para. 2). Wang teaches a method comprising “genetic perturbations” (Pg. 10850, Col. 1, Para. 1). Thus, Wang teaches a method comprising step (a)(i), providing a plurality of cells, wherein the plurality of cells comprise at least one cell comprising at least one genetic perturbation, wherein said at least one cell comprising the at least one genetic perturbation comprises a barcode sequence associated with the genetic perturbation.
Regarding claim 1, Wang teaches a method comprising “the samples were equilibrated in 30% formamide in 2x SSC for 5 minutes before FISH staining. The FISH hybridization buffer contains 30% formamide (ThermoFisher, AM9342), 60% stellaris RNA FISH hybridization buffer (Biosearch, SMF-HB1-10), 10% 25 mg/mL Yeast tRNA and 1:100 murine RNase inhibitor. The samples were stained with 300 nM FISH probes for the reporter gene, 300 nM FISH probes for RNA phenotype (i.e., 6 RNA species) imaging, and 100 nM primary amplification probes for barcode imaging at 37 ℃ overnight.” (Supplement Pg. 5, Imaging sample Preparation, Para. 1).
Wang teaches a method comprising “Then the samples were washed in 30% formamide in 2x SSC twice and stained with 100 nM secondary amplification probes for barcode imaging in 10% hybridization buffer (10% formamide, 80% stellaris RNA FISH hybridization buffer, 10% 25 mg/mL Yeast tRNA and 1:100 murine RNase inhibitor) for an hour at 37 ℃.”(Supplement Pg. 6, Imaging sample Preparation, Para. 1). “formamide” reads on preserving the barcode sequence. “stellaris RNA FISH hybridization buffer” reads on preserving the barcode sequence. “RNase inhibitor” on reads on preserving the barcode sequence. Thus, Wang teaches a method comprising step (a)(ii), preserving the barcode sequence of the at least one cell before completing (b).
Regarding claim 1, Wang teaches a method comprising “For each round of hybridization,
fluorescence signals … were imaged. After each round, the dyes on the readout probes were cleaved … followed by hybridization of the readout probes for next round.” (Supplement Pg. 8, Imaging sample Preparation, Para. 1). Wang teaches a method comprising “all-imaging–based pooled-library CRISPR screening in mammalian cells. This approach allows both high-content phenotype imaging of multiple molecular targets in individual cells and high-accuracy identification of the genotype of each cell, the latter achieved by associating each sgRNA with unique barcodes and reading out the barcodes using multiplexed error-robust fluorescence in situ hybridization (MERFISH)” (Pg. 10842, Col. 2, Para. 2). Wang teaches a method comprising “detected the reporter gene mRNA with single molecule fluorescence in situ hybridization (smFISH), so that only the barcode signals that colocalized with the reporter gene signals were considered (Fig. 1A)” (Pg. 10844, Col. 1, Para. 2). Thus, Wang teaches a method comprising step (b), performing at least one round of fluorescence in situ hybridization (FISH), wherein the at least one round of FISH comprises (i) detecting a fluorescent oligonucleotide uniquely associated with an mRNA transcript, or (ii) detecting a fluorescent oligonucleotide uniquely associated with a protein of interest.
Regarding claim 1, Wang teaches a method comprising “all-imaging–based pooled-library CRISPR screening in mammalian cells” (Pg. 10842, Col. 2, Para. 2). Wang teaches a method comprising “The library was delivered into the genome of U-2 OS cells… most transfected cells received only one barcode. We then measured the barcode signals for individual cells using the multiplexed detection scheme as described above. After each round of hybridization, we observed clear barcode signals colocalizing with the smFISH signals of the reporter gene (luciferase-mCherry) mRNA (Fig. 1C)” (Pg. 10844, Col. 1 Para. 3; Fig. 1C). Wang teaches a method comprising “barcode signal is amplified” (Fig. 1a legend). Wang teaches a method comprising “deduce the barcode identity of that cell from the sequencing results” (Pg. 10844, Col 2, Para. 3). “U-2 OS cells” read on human cells. Thus, Wang teaches a method comprising step (c), performing pooled optical screening in human cells (POSH), comprising amplifying the barcode in the at least one cell and sequencing.
Wang does not explicitly teach amplifying the barcode sequence and sequencing the barcode sequence in situ.
Feldman discloses “Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells and accurate sequence reads sufficient to distinguish >106 perturbations. In situ amplification takes ~2 d, while sequencing can be performed in ~1.5 h per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.” (Abstract).
Regarding claim 1, Feldman teaches a method wherein “a population of cells is subjected to a library of genetic perturbations” (Figure 1a legend). Feldman teaches a method wherein “genotype is linked to phenotype at single-cell resolution by sequencing cellular perturbation identity in situ, within fixed cells. Perturbation identities are deduced from mRNA containing either the single-guide RNA (sgRNA) sequence itself or a short barcode, analogous to barcode capture in pooled single-cell RNA sequencing screens. Barcodes are read out in fixed cells via padlock-based in situ sequencing, a process involving padlock probe hybridization and gap filling, rolling circle amplification (RCA), and in situ sequencing by synthesis (SBS) (Fig. 2).” (Pg. 478, Development of optical pooled screens, Para.1). Furthermore, Feldman teaches a method wherein “Several recent technologies enable pooled imaging screens in bacterial and mammalian systems based on in situ optical barcoding of genetic perturbations… Barcoding
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methods have relied on in situ sequencing, as in the optical pooled screening approach presented here, or iterative fluorescence in situ hybridization (FISH) to map genetic perturbations”, “in situ sequencing achieves higher imaging throughput and direct detection of CRISPR sgRNAs using the standard CROP-seq vector” and FISH provides higher detection sensitivity, which may be useful in mammalian cell types” (Pg. 478, Comparison with alternative methods, Para.1). Thus, Wang and Feldman suggest a method comprising step (c) performing pooled optical screening in human cells (POSH), comprising amplifying the barcode sequence in the at least one cell and sequencing the barcode sequence in situ.
Wang and Feldman are both considered to be analogous to the claimed invention because they are in the same field of pooled genetic perturbation screens with image-based phenotypes. Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date to modify the method according to the instant application claim limitations of claim 1 steps (a)-(b) and wherein step (c) comprises performing pooled optical screening in human cells (POSH), comprising amplifying the barcode in the at least one cell and sequencing the barcode sequence as taught by Wang to incorporate a method of amplifying the barcode sequence and sequencing the barcode sequence in situ as taught by Feldman because these claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome of a method according to the limitations of the instant application claim 1 steps (a) – (c). A person of ordinary skill in the art would have had a reasonable expectation of success in incorporating the amplification of the barcode sequence and in situ sequencing because the method of Wang teaches amplifying the barcode and decoding the barcode to deduce the barcode identity of that cell from the sequencing results, Feldman suggests that in situ sequencing achieves higher imaging throughput and both Wang and Feldman teach performing pooled optical screening in human cells. The skilled artisan would have been motivated to incorporate barcode sequence amplification and in situ sequencing because doing so would allow for sequencing verification in the spatial context of the human cells.
The teachings of Wang and Feldman are documented above in the rejection of claim 1 under 35 U.S.C. 103. Claims 2, 7, 13, 17, 25, 27, 61-62, 77-78, 82, 97, 99-101 and 147-148 depend on claim 1. Claims 62 and 77 depend on claim 61, which depends on claim 1. Claim 80 depends on claim 32, which depends on claim 1. Claim 82 depends on claim 62, which depends on claim 61, which depends on claim 1. Claim 78 depends on claim 77, which depends on claim 61, which depends on claim 1. Claim 100 depends on claim 99, which depends on claim 97, which depends on claim 2, which depends on claim 1.
Regarding claim 2, Wang teaches a method further comprising” imaged the phenotype of each cell … using immunofluorescence (Fig. 2B)” (Pg. 10844 Col 2 Para. 3) and “phenotype imaging” (Supplement Pg. 5, Imaging sample preparation, Para.1). Wang teaches a method further comprising “DAPI staining was imaged and used for cell segmentation and nucleus identification” (Supplement Pg. 8, Para.3) and “cell boundary identification” (Supplement Pg. 9, Para.1). Thus, Wang and Feldman suggest a method further comprising analyzing the phenotype of the at least one cell; wherein analyzing the phenotype comprises at least one assay selected from the group consisting of label-free imaging, high content imaging, calcium imaging, immunohistochemistry, cell morphology imaging, protein aggregation imaging, cell-cell interaction imaging, live cell imaging, and any other imaging-based assay modality.
Regarding claims 7 and 13, Wang teach a method wherein “pooled-library CRISPR screening in mammalian cells” (Pg. 10842, Col. 2, Para. 2). Thus, Wang and Feldman suggest a method wherein the at least one cell comprises a CRISPR system; and wherein the at least one cell has been contacted with a gRNA to generate the at least one perturbation.
Regarding claim 17, Wang teaches “sgRNA-reporter-barcode libraries were performed in pooled manner… These libraries were cloned into the lentiviral vector pFUGW… The lentiviral libraries were used to infect the U-2 OS cells” (Pg. 10850, Materials and methods, Para. 2-3). Wang teaches “a population of cells is subjected to a library of genetic perturbations, such as guide RNAs for CRISPR screens” (Figure 1a legend; Fig. 1). Thus, Wang and Feldman suggest a method further comprising contacting the plurality of cells with a gRNA library comprising a plurality of different gRNAs to generate a plurality of genetic perturbations comprising the at least one genetic perturbation.
Regarding claim 25, “cells were fixed” Pg. 10850, Materials and methods, Para. 3).
Wang teaches a method wherein “samples were… fixed with 4% PFA for 30 minutes… before FISH staining. … The samples were stained with 300 nM FISH probes” (Supplement Pg. 5, Imaging sample preparation, Para. 1). Thus, Wang and Feldman suggest a method further comprising fixing the plurality of cells on a surface prior to step (b).
Regarding claim 27, Wang teaches a method wherein “samples were … permeabilized” (Supplement Pg. 5, Imaging sample preparation, Para. 1). Thus, Wang and Feldman suggest a method further comprising permeabilizing the plurality of cells.
Regarding claims 61-62,and 77, Feldman teaches a method wherein “Barcodes are read out in fixed cells via padlock-based in situ sequencing, a process involving padlock probe hybridization and gap-filling, rolling circle amplification (RCA), and in situ sequencing by synthesis (SBS) (Fig. 2). We carefully optimized the in situ sequencing protocol in adherent cells, adding glutaraldehyde fixation after reverse transcription of cDNA and maximizing gap-fill reaction efficiency by titrating dNTP concentration. These optimizations improve both the number and brightness of sequencing reads, enabling high-throughput optical pooled screening with perturbations successfully identified for a large fraction of cells when sequenced with 10X magnification” (Pg 478, Development of optical pooled screens, Para. 1; Figure 2b see below). Figure 2B above depicts steps (A)-(C) of claim 61(Figure 2B, steps Expression of sgRNA to Gap-fill), step (D) of claim 62 (Figure 2B, step RCA), and further comprising fixing the reverse transcribed barcode sequence in place after step (a) of POSH. (Figure 2B, steps Expression of sgRNA to RT).
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Thus, Wang and Feldman suggest a method, wherein POSH comprises:(A) reverse transcribing the barcode sequence to form a reverse transcribed barcode sequence; (B) hybridizing at least one padlock probe to the reverse transcribed barcode sequence, wherein: the at least one padlock probe comprises a first barcode hybridization sequence and a second barcode hybridization sequence, the reverse transcribed barcode sequence comprises a first padlock probe hybridization sequence and a second padlock probe hybridization sequence flanking a target sequence, and the first barcode hybridization sequence hybridizes with first padlock probe hybridization sequence and the second barcode hybridization sequence hybridizes with second padlock probe hybridization sequence; and (C) connecting the ends of the at least one padlock probe to form a circular probe; further comprising (D) forming a barcode amplicon using the circular probe as a template, wherein the barcode amplicon comprises a plurality of copies of the barcode sequence; and further comprising fixing the reverse transcribed barcode sequence in place after step (a) of POSH.
Regarding claim 78, Feldman teaches method wherein “adding glutaraldehyde fixation after reverse transcription” (Pg 478, Development of optical pooled screens, Para. 1; Figure 2b see above).” Feldman teaches a method wherein “fixation with freshly diluted 3% PFA + 0.1% glutaraldehyde in PBS (post-fixation solution)” (Pg. 498, Step 97). Thus, Wang and Feldman suggest a method wherein fixing the reverse transcribed barcode sequence comprises at least one of paraformaldehyde (PFA) or glutaraldehyde treatment.
Regarding claim 82, Wang teaches a method comprising at least one round of RNA FISH as discussed above. Feldman teaches a method wherein “rolling circle amplification (RCA)” of the padlock probe to form copies of the barcode. (Pg. 482, Box 3; Pg 478, Development of optical pooled screens, Para. 1). Thus, Wang and Feldman suggest a method wherein the barcode amplicon is formed after the at least one round of RNA FISH.
Regarding claim 97, Feldman depicts an with image based phenotyping before screen analysis. (Figure 2) Feldman teaches a method wherein “A live-cell or fixed-cell imaging assay is performed to generate an optical phenotypic profile of individual cells” (Fig. 2 legend), and “Image-based screens can be performed with live-cell or fixed-cell phenotyping. Presented below are examples of both live-cell (Step 88) and fixed-cell (Step 99) phenotyping protocols for a screen measuring p65 translocation in HeLa cells.” (Steps 85–93, image-based phenotyping; Pg. 502, Analysis Step 133). Thus, Wang and Feldman suggest a method wherein analyzing the phenotype of the at least one cell is performed before segmenting a morphological image of the cell, wherein segmenting comprises detecting the cell and identifying the boundaries of the cell in the morphological image.
Regarding claim 99, Feldman teaches a method wherein “phenotyping data (p65 localization) are shown for a single field of view. White outlines in sequencing images represent individual cells; colored outlines in the phenotype image represent clusters of neighboring cells with identical sgRNA assignments (Figure 4a; Pg. 482). Wang teaches Figure 5C which depicts processed images of the same cell wherein “MALAT1 staining is shown in magenta, and SON staining is shown in green” (Figure 5C). Thus, Wang and Feldman suggest a method further comprising processing and/or transforming the morphological image to obtain images of the same cell with different readouts.
Regarding claim 100, Feldman teaches Figure 4A which depicts images of the same cell for at least one round of FISH and barcode decoding (green box) and phenotype imaging (pink
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and green box). (Figure 4A see below) Thus, Wang and Feldman suggest a method wherein the images of the same cell are obtained from the at least one round of FISH, sequencing of the barcode sequence in situ, and/or segmenting of the morphological image of the cell.
Regarding claim 101, Wang teaches Figure 2B which depicts “Images showing HA and Myc immunostaining signals in two different channels. The nuclei with strong HA signals have weak Myc signals and vice versa. The cell boundaries are labeled in green. The nucleus boundaries of HA expression cells and Myc-expressing cells are labeled in red and blue, respectively” (Figure 2B). Thus, Wang and Feldman suggest a method further comprising: receiving a first image depicting the plurality of cells, wherein the first image indicates each cell of the plurality of cells by a boundary and associates each cell of the plurality of cells with a corresponding cell identifier.
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Regarding claim 101, Wang teaches Figure 1C which depicts “(C, Upper) Example images showing reporter mRNA smFISH signal (green) and the signals for each of the three trit values (0, 1, and 2) for a single trit in the barcode (magenta). (C, Lower) Enlarged views of the white-boxed region in the upper images, with the reporter gene signal shown on the left and the overlay between the reporter gene signal and the barcode trit signals on the right. Trit value 1 has a high colocalization ratio for this cell, whereas Trit values 0 and 2 do not have high colocalization ratios” (Figure 1C and legend). Wang also teaches a method comprising “We then measured the barcode signals for individual cells using the multiplexed detection scheme as described above. After each round of hybridization, we observed clear barcode signals colocalizing with the smFISH signals of the reporter gene (luciferase-mCherry) mRNA (Fig. 1C). For each trit detection, three trit values were separately probed (in different pseudocolor channels as described earlier), and three distinct populations of cells were observed, representing cells expressing barcodes with three different trit values (Fig. 1D and SI Appendix, Fig. S2). We used a k-means clustering algorithm to separate the three populations of cells, and a trit value assigned to each population based on which of the three pseudocolor channels assigned to this trit exhibited the highest fraction of reporter gene mRNA spots that were colocalized to the trit signal. The detection of 12 trits using 36 pseudocolor channels allowed us to assign a barcode to each cell. The decoded barcodes for the majority (∼57%) of cells matched the ∼2,000 barcodes in the library determined by sequencing” (Pg. 10844, Col. 1, Para. 3-4). Thus, Wang and Feldman suggest a method further comprising receiving a second image depicting locations of a plurality of barcode sequences, wherein the plurality of barcode sequences are associated with the plurality of cells after POSH is performed; aligning the first image and the second image; based on the alignment of the first image and the second image, identifying an association between each cell of the plurality of cells and a corresponding barcode sequence of the plurality of barcode sequences; receiving a FISH image of the plurality of cells after the at least one round of FISH is performed on the plurality of cells; aligning the first image and the FISH image; based on the alignment between the first image and the FISH image, resizing the FISH image; associating each cell of the plurality of cells with a portion of the resized FISH image, the corresponding barcode sequence, and the corresponding cell identifier; and analyzing known phenotypes or identifying new phenotypes of the plurality of cells.
Regarding claims 147-148, Feldman teaches method wherein “adding glutaraldehyde fixation after reverse transcription” (Pg 478, Development of optical pooled screens, Para. 1; Figure 2b see below) Thus, Wang and Feldman suggest a method wherein preserving the barcode sequence of the at least one cell comprises reverse transcribing the barcode sequence of the at least one cell; and wherein preserving the barcode sequence of the at least one cell further comprises fixing the reverse transcribed barcode sequence of the at least one cell.
Response to Arguments
Applicants’ arguments filed 03/04/2026 (Pg.12-13) with respect to the rejection of claims 1-2,7,13,17,25,27,30,32,34,46,57,61-62,77-78,80,82,86-87,89,97,99-101 and 147-148 under 35 U.S.C. 102 have been fully considered and but do not apply to the new grounds of rejection under 35 U.S.C. 103 in view Wang and Feldman. As necessitated by claim amendments, the amended claims required a new grounds of rejection. To clarify some instances argued that may still apply in the response filed 03/04/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “. The Feldman reference indicates that it was published in February 2022 but does not provide an exact publication date. Applicant requests that the Examiner establish that Feldman is prior art under 35 U.S.C. 102 (a)(l) by providing authoritative evidence that Feldman was published prior to February 23, 2022.” (Pg. 17).
Response: In response to applicant's arguments against the date of publication for the Feldman reference. The full date of the first publicly available version of the Feldman et al. (2022) article is Jan. 12, 2022 and now reflected in the new grounds of 35 U.S.C. 103 rejections above for clarity.
Claims 30, 32, 34, 46 and 80 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. “Wang”; (2019). Proceedings of the National Academy of Sciences of the United States of America (PNAS), 116(22), 10842–10851., Published online May 13, 2019) in view of Feldman et al. “Feldman”; (2022). Pooled genetic perturbation screens with image-based phenotypes. Nature protocols, 17(2), 476–512., Published online Jan. 12, 2022) as applied to claims 1-2, 7, 13, 17, 25, 27, 61-62, 77-78, 82, 97, 99-101 and 147-148 above, and further in view of Samusik et al. (“Samusik”; Patent App. Pub. US 20190055594 A1, Feb. 21, 2019).
The teachings of Wang and Feldman are documented above in the rejection of claims 1-2, 7, 13, 17, 25, 27, 61-62, 77-78, 82, 97, 99-101 and 147-148 under 35 U.S.C. 103. Claims 32, 34 and 46 depend on claim 30, which depends on claim 1. Wang teaches “This screening method should be broadly applicable to interrogating genetic factors controlling or regulating a broad spectrum of phenotypes, including morphological features, molecular organizations, and dynamics of cellular structures, as well as cell–cell interactions. We also anticipate that this screening approach can be combined with highly multiplexed DNA, RNA, and protein imaging approaches, including genomic-scale imaging approaches, to profile factors involved in gene regulation and other genomic functions in a high-throughput manner” (Pg. 10850, Discussion, Last para.). Thus, one of ordinary skill in the art would be motivated to modify the method of Wang and Feldman to incorporate further aspects of the field. Feldman does not explicitly teach the limitations of claims 30, 32, 34, 46 and 80.
Samusik discloses “SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer.” (Abstract).
Regarding claims 30 and 32, Samusik teaches a method wherein “a simple two-probe proximity ligation system termed SNAIL-RCA that enables in situ amplification, detection and visualization of genes. RCA products are detected via hybridization of unlabeled detection probes coupled with single-nucleotide extension with fluorescent nucleotide analogs. Fluorescent imaging and automatic image analysis enable precise quantification of expression levels. Multiplexing is enabled through re-hybridization, which, combined with parse barcoding strategy enables simultaneous detection of hundreds or thousands of genes. We show that SNAIL enables detection of single-cell transcription heterogeneity in cell cultures as well as tissue samples” (Para. 146; Para.147-149; Fig. 1). A person of ordinary skill in the field would know to design the SNAIL probes to hybridize to the preferred 3’ or 5’ end of the mRNA transcript. Thus, Wang, Feldman and Samusik suggest a method wherein the at least one round of FISH comprises at least one round of RNA FISH, wherein each round of RNA FISH is uniquely associated with at least one mRNA transcript from the at least one cell, wherein the at least one round of RNA FISH comprises:(i) contacting a plurality of mRNA transcripts comprising the at least one mRNA transcript with a plurality of 3' or 5’ loop probes, wherein each 3' or 5’ loop probe comprises a first target hybridization sequence complementary to a first portion of an mRNA transcript of the plurality of mRNA transcripts;(ii) contacting the plurality of mRNA transcripts comprising the at least one mRNA transcript with a plurality of 5' or 3’ probes, wherein each 5' or 3’ probe comprises a second target hybridization sequence complementary to a second portion of the mRNA transcript of the plurality of mRNA transcripts, wherein each 5' or 3’ probe is capable of specifically hybridizing with a 3' or 5’ loop probe, wherein hybridization of a 5' or 3’ probe with a corresponding 3' or 5’ loop probe forms a loop in the 3' or 5’ loop probe:(iii) connecting the ends of the loop in each 3' or 5’ loop probe to form a plurality of circular probes:(iv) amplifying a plurality of target sequences using the circular probes as templates to form a plurality of DNA amplicons; and(v) detecting the DNA amplicons by the at least one round of RNA FISH.
Wang, Feldman and Samusik are considered to be analogous to the claimed invention because they are in the same field of barcode identification for detection of genetic target nucleic acid in situ. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of pooled optical screening of genetically barcoded cells comprising genetic perturbations and simultaneous transcriptional measurements as taught by Wang and Feldman to incorporate the method of RNA FISH according to the limitations of claims 30 or 32, as taught by Samusik and provide method for performing at least one round of RNA FISH, contacting mRNA transcripts with a 3’ or 5’ loop probe, respectively, targeting a first portion of the mRNA transcript, then contacting mRNA transcripts with a 5’ or 3’ loop probe, respectively, targeting a second portion of the mRNA transcript, wherein the 5’ and 3’ loop probe are capable hybridization to each other, circularizing the 3’ loop or 5’ loop, respectively, amplification of target sequence using circular probe to form a plurality of amplicons and detection of amplicons by at least one round of FISH. Doing so would provide improved sensitivity and reduce variability.
Regarding claim 34, Samusik teaches a method wherein “RCA product can be detected by various methods, which include, without limitation, hybridization to a sequence specific detection oligonucleotide (DO), also referred to as a detection probe. In some embodiments the DO is conjugated to a detectable label, e.g. fluorophore, lanthanide, biotin, radionuclide, etc., where the label may be detectable by optical microscopy, SIMS ion beam imaging, etc. … the presence of the DO can be detected in a polymerization reaction primed by the DO, and where the polymerization reaction may comprise one or more dNTP comprising a detectable label. Such polymerization products may further comprise a step of adding a label, detecting a label, and removing the label for sequential detection of different products. The detection primer can be specific for a region of the RCA amplification product that is specific for the target gene,” (Para. 19-20; Para.146). Thus, Wang, Feldman and Samusik suggest a method wherein detecting the DNA amplicons comprises at least one of: (I) labeling the DNA amplicons with a fluorophore, an isotope, a mass tag, or a combination thereof; (II) hybridizing an adapter oligonucleotide to the DNA amplicon; (III) hybridizing a detection probe to the adapter oligonucleotide, (IV) hybridizing a detection probe to the adapter oligonucleotide, wherein the detection probe comprises a fluorophore, an isotope, a mass tag, an oligonucleotide, or a combination thereof; or (V) imaging the DNA amplicons.
Regarding claim 46, Samusik teaches a method wherein “RCA products” (Para.146; Figure 1B). Thus, Wang, Feldman and Samusik suggest a method wherein the DNA amplicon is formed using rolling circle amplification (RCA).
Regarding claim 80, Samusik teaches a method wherein “Both enzymes were used according to manufacturers' instructions, with the addition of 40 U/mL RNasin” (Para. 151). Furthermore, Wang teaches a method comprising “Then the samples were washed in 30% formamide in 2x SSC twice and stained with 100 nM secondary amplification probes for barcode imaging in 10% hybridization buffer (10% formamide, 80% stellaris RNA FISH hybridization buffer, 10% 25 mg/mL Yeast tRNA and 1:100 murine RNase inhibitor) for an hour at 37 ℃… The samples labeled with FISH probes for phenotype imaging and reporter gene mRNA imaging, and primary and secondary amplification probes for barcode imaging were washed twice in 30% formamide in 2x SSC, and then embedded in 4% polyacrylamide gel… The FISH probes… were conjugated with acrydite, which can crosslink to the polyacrylamide gel and retain these probes as well as their bound RNA within the gel” (Supplement Pg. 6, Imaging sample Preparation, Para. 1). “ formamide” reads on preserving the barcode sequence. “stellaris RNA FISH hybridization buffer” reads on preserving the barcode sequence. “RNase inhibitor” on reads on preserving the barcode sequence. “embedded in 4% polyacrylamide gel” reads on preserving the barcode sequence. “conjugated with acrydite, which can crosslink to the polyacrylamide gel” reads on preserving the barcode sequence. Thus, Wang, Feldman and Samusik suggest a method wherein (a)(ii) is performed after step (iv) and before step (v) of the at least one round of RNA FISH.
Response to Arguments
Applicants’ arguments filed 03/04/2026 (Pg.14) with respect to the rejection of claims 1-30, 32,34 and 46 under 35 U.S.C. 103 have been fully considered and but do not apply to the new grounds of rejection under 35 U.S.C. 103 in view Wang, Feldman and Samusik. As necessitated by claim amendments, the amended claims required a new grounds of rejection.
Claims 57 and 86 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. “Wang”; (2019). Proceedings of the National Academy of Sciences of the United States of America (PNAS), 116(22), 10842–10851., Published online May 13, 2019) in view of Feldman et al. “Feldman”; (2022). Pooled genetic perturbation screens with image-based phenotypes. Nature protocols, 17(2), 476–512., Published online Jan. 12, 2022) as applied to claims 1-2, 7, 13, 17, 25, 27, 61-62, 77-78, 82, 97, 99-101 and 147-148 above, and further in view of Urbanek et al. (“Urbanek”; (2016). RNA FISH for detecting expanded repeats in human diseases. Methods, 98, 115-123.).
The teachings of Wang and Feldman are documented above in the rejection of claims 1-2, 7, 13, 17, 25, 27, 61-62, 77-78, 82, 97, 99-101 and 147-148 under 35 U.S.C. 103. Claim 86 depends on claim 57 which depends on claim 1. Wang teaches “This screening method should be broadly applicable to interrogating genetic factors controlling or regulating a broad spectrum of phenotypes, including morphological features, molecular organizations, and dynamics of cellular structures, as well as cell–cell interactions. We also anticipate that this screening approach can be combined with highly multiplexed DNA, RNA, and protein imaging approaches, including genomic-scale imaging approaches, to profile factors involved in gene regulation and other genomic functions in a high-throughput manner” (Pg. 10850, Discussion, Last para.). Thus, one of ordinary skill in the art would be motivated to modify the method of Wang and Feldman to incorporate further aspects of the field. Feldman does not explicitly teach the limitations of claims 57 and 86.
Urbanek discloses “RNA fluorescence in situ hybridization (FISH) is a widely used technique for detecting transcripts in fixed cells and tissues. Many variants of RNA FISH have been proposed to increase signal strength, resolution and target specificity. The current variants of this technique facilitate the detection of the subcellular localization of transcripts at a single molecule level. Among the applications of RNA FISH are studies on nuclear RNA foci in diseases resulting from the expansion of tri-, tetra-, penta- and hexanucleotide repeats present in different single genes. The partial or complete retention of mutant transcripts forming RNA aggregates within the nucleoplasm has been shown in multiple cellular disease models and in the tissues of patients affected with these atypical mutations… In this article, we summarize the results obtained with FISH to examine RNA nuclear inclusions. We provide a detailed protocol for detecting RNAs containing expanded CAG and CUG repeats in different cellular models, including fibroblasts, lymphoblasts, induced pluripotent stem cells and murine and human neuronal progenitors. We also present the results of the first single-molecule FISH application in a cellular model of polyglutamine disease.” (Abstract).
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Regarding claim 57, Urbanek teaches a method wherein “imaging RNA foci with FISH techniques” (Pg. 116, Col. 1, 1.2. RNA foci in repeat expansion diseases, Para.1). Urbanek teaches a method wherein “RNAs were detected with LNA (Exiqon; MA, USA), RNA (IDT; IA, USA) or DNA probes (IBB PAN, oligo.pl; Poland)” (Pg. 116, Col. 2, 2.3 FISH probes, Para.1). Urbanek teaches a method wherein “Typically, in RNA FISH for the detection of mutant transcripts with expanded repeats, probes targeting the repeated sequence are used.” (Pg. 117, Col. 1, 3.2 Probes, Para. 1; Figure 1, see below). Thus, Wang, Feldman and Urbanek suggest a method wherein the detecting the fluorescent oligonucleotide uniquely associated with the mRNA transcript comprises:(i) contacting RNA foci with a fluorescent oligonucleotide probe, wherein each fluorescent oligonucleotide probe comprises a target hybridization sequence complementary to a portion of a sequence in the RNA foci; and (ii) detecting the fluorescent oligonucleotide probe by imaging.
Regarding claim 86, Urbanek teaches a method wherein “overnight at an optimal temperature. The hybridization temperature should be experimentally determined for every probe.” (Pg. 118, Col.2, 3.4. Hybridization, Para. 1). Of note, "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). Thus, Wang, Feldman and Urbanek suggest a method wherein step (i) of contacting RNA foci with the fluorescent oligonucleotide probe is carried out during an incubation period at a temperature of at least 50 °C.
Wang, Feldman and Urbanek are considered to be analogous to the claimed invention because they are in the same field of detection of genetic target in situ. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of pooled optical screening of genetically barcoded cells comprising genetic perturbations and simultaneous transcriptional measurements as taught by Wang and Feldman to incorporate the method of RNA foci FISH according to the limitations of claims 57 and 86 and wherein the incubation of the fluorescent probe with RNA foci is carried out at an optimal temperature of 50 °C as taught by Urbanek and provide method for performing at least one round of RNA foci FISH. Doing so would enable qualitative and quantitative analysis of RNA molecules and the characterization of nuclear foci.
Response to Arguments
Applicants’ arguments filed 03/04/2026 (Pg.14) with respect to the rejection of claims 1, 57 and 86 under 35 U.S.C. 103 have been fully considered and but do not apply to the new grounds of rejection under 35 U.S.C. 103 in view Wang, Feldman and Urbanek. As necessitated by claim amendments, the amended claims required a new grounds of rejection. To clarify some instances argued that may still apply in the response filed 03/04/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “. Urbanek is not pertinent to the claimed invention. It addresses: (a) disease-specific RNA foci (pathological aggregates), (b) repeat expansion sequences (CAG, CUG, CCUG, GGGGCC), and (c) diagnostic applications in patient tissues. Urbanek is not reasonably pertinent to pooled optical screening of, e.g., CRISPR perturbations in healthy cells.” (Pg. 15).
Response: In response to applicant's argument that Urbanek is nonanalogous art, it has been held that a prior art reference must either be in the field of the inventor’s endeavor or, if not, then be reasonably pertinent to the particular problem with which the inventor was concerned, in order to be relied upon as a basis for rejection of the claimed invention. See In re Oetiker, 977 F.2d 1443, 24 USPQ2d 1443 (Fed. Cir. 1992). In this case, Urbanek is in the field of methods of identifying amplified genetic perturbations and phenotypic characteristics in human cells using RNA foci FISH. The instant invention Thus, Urbanek is reasonably pertinent to pooled optical screening of, e.g., CRISPR perturbations in healthy cells.
Claims 87 and 89 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. “Wang”; (2019). Proceedings of the National Academy of Sciences of the United States of America (PNAS), 116(22), 10842–10851., Published online May 13, 2019) in view of Feldman et al. “Feldman”; (2022). Pooled genetic perturbation screens with image-based phenotypes. Nature protocols, 17(2), 476–512., Published online Jan. 12, 2022) as applied to of claims 1-2, 7, 13, 17, 25, 27, 61-62, 77-78, 82, 97, 99-101 and 147-148 above, and further in view of Regev et al. (“Regev”; Patent App. Pub. WO 2020160044 A1, Aug. 08, 2020).
The teachings of Wang and Feldman are documented above in the rejection of claims 1-2, 7, 13, 17, 25, 27, 61-62, 77-78, 82, 97, 99-101 and 147-148 under 35 U.S.C. 103. Claims 87 and 89 depend on claim 1. Wang teaches a method wherein “phenotype imaging a nuclear speckle protein, SON, using immunolabeling with an oligonucleotide-conjugated antibody” (Pg. 10848, Col. 1, Para. 1) and “The samples were washed three times with 1xPBS and incubated with 1:300 oligonucleotide-labeled secondary antibody for one hour. The oligonucleotide-labeled secondary antibody can be later probed by readout probes with sequence complementary to the oligonucleotide sequence on the antibody.” (Supplement Pg. 5, Imaging sample preparation, Para. 1). Feldman does not explicitly teach the limitations of claims 87 and 89.
Regev discloses “The present invention provides methods and tools for analyzing genetic interactions. The subject matter is generally directed to single-cell genomics and proteomics, including methods of performing genome-wide CRISPR perturbation screens and determining gene expression phenotypes.” (Abstract)
Regarding claim 87 and 89, Regev teaches a method wherein “Capture molecules include molecules such as … DNA segments … antibodies, tailored for the molecules of interest. In embodiments, the capture molecule comprises a sequence specific for a target molecule of interest, a sequence specific for capture of an SNP … an antibody, …or a combination thereof.” (Para. 127). Regev teaches a method wherein “the spatial barcodes further comprise a capture molecule or moiety” and “ a sequence complementary to a second nucleotide sequence which allows for ligation of the spatial barcode to another entity comprising the second nucleotide sequence, e.g., another detectable oligonucleotide tag or an oligonucleotide adapter. A priming sequence is a sequence complementary to a primer, e.g., an oligonucleotide primer used for an amplification reaction such as but not limited to PCR” (Para. 128; Para. 129). Regev teaches a method wherein “ tracr and tracr mate sequences” and “Suitable attachments include any moiety that can be added to the linker to add additional properties to the linker, such as but not limited to, fluorescent labels “(Para. 225) ). Regev teaches a method wherein “imaging systems as needed“ (Para. 131). Furthermore, Regev teaches a method wherein “direct and secondary antibody fluorescent staining can be utilized to sense proteins in the tissue sample. Embodiments may comprise DNA-barcode antibodies” (Para. 198). Thus, Wang, Feldman and Regev suggest a method wherein the at least one round of FISH comprises at least one round of immunoFISH, wherein each round of immunoFISH is uniquely associated with at least one protein of interest from the at least one cell, wherein the at least one round of immunoFISH comprises:(i) contacting the at least one cell with a conjugate, the conjugate comprising an antibody which specifically binds the at least one protein of interest and a DNA oligonucleotide comprising a protein identification sequence uniquely associated with the protein of interest;(ii) contacting the conjugate with an adapter oligonucleotide, wherein the adapter oligonucleotide comprises a first adapter sequence complementary to a portion of the DNA oligonucleotide:(iii) contacting the adapter oligonucleotide with a fluorescent oligonucleotide capable of hybridizing with a second adapter sequence of the adapter oligonucleotide;(iv) imaging the at least one cell to detect the at least one fluorescent oligonucleotide; and wherein (i) contacting the at least one cell with a first antibody which specifically binds the at least one protein of interest; (ii) contacting the antibody with a conjugate comprising an antibody which specifically binds the first antibody and a DNA oligonucleotide comprising a protein identification sequence uniquely associated with the protein of interest; (iii) contacting the conjugate with an adapter oligonucleotide, wherein the adapter oligonucleotide comprises a first adapter sequence complementary to a portion of the DNA oligonucleotide; (iv) contacting the adapter oligonucleotide with a fluorescent oligonucleotide capable of hybridizing with a second adapter sequence of the adapter oligonucleotide; (v) imaging the at least one cell to detect the at least one fluorescent oligonucleotide.
Wang, Feldman and Regev are both considered to be analogous to the claimed invention because they are in the same field of preparing nucleic acids for analysis. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of pooled optical screening of genetically barcoded cells comprising genetic perturbations and simultaneous transcriptional measurements as taught by Feldman to incorporate the method of immunoFISH according to the limitations of claims 87 or 89 as taught by Regev and provide method for performing at least one round of immunoFISH. Doing so would allow for various antibody-based phenotypic readouts from the pooled optical screening.
Response to Arguments
Applicants’ arguments filed 03/04/2026 (Pg.14) with respect to the rejection of claims 1, 57 and 86 under 35 U.S.C. 103 have been fully considered and but do not apply to the new grounds of rejection under 35 U.S.C. 103 in view Wang, Feldman and Regev. As necessitated by claim amendments, the amended claims required a new grounds of rejection.
Conclusion of Response to Arguments
In view of the amendments, revised and new grounds of rejections, responses to arguments are documented in this Final Office Action. No claims are in condition for allowance.
Conclusion
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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