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 .
Status of the Claims
Claims 1, 6-15, and 17-22 are pending. Claims 1, 6-15, and 17-22 are the subject of this Non-Final Office Action. Claims 3-5, 16, and 23 have been cancelled.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 02/18/2026 has been entered.
New Grounds - Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 6, 9, 12, 17, and 19-20 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.
Claims 6, 9, 12, 17, and 19-20 depend upon claims which have been cancelled. It is unclear what the full scope of the claims encompass as the parent claims have been cancelled.
New Grounds - 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.
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.
Claim(s) 1, 6, 9-15, and 17-21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Datlinger et al. (Pooled CRISPR screening with single-cell transcriptome readout. Nat. Methods, 4, 2017, cited in the IDS filed 11/02/2022; previously cited) in view of Meltzer et al. (WO 2020/069298 A1, cited in the IDS filed 11/02/2022; previously cited) and Regev et al. (US 2020/0362334 A1; previously cited), and evidenced by Macosko et al. (Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell, 161, 2015; previously cited) and Hatori et al. (Particle-templated emulsification for microfluidics – free digital biology. Analytical Chemistry, 90, 2018, 9813-9820; cited on the IDS filed 11/02/2022; previously cited).
Regarding claim 1, Datlinger teaches a method of screening single-cell where the cells comprise polyadenylated guide RNAs (pg. 297, right col., par. 2). A barcoded bead and a cell are encapsulated within a partition (Fig. 1c). The cells are then lysed, allowing the polyadenylated guide RNAs to be indexed (Fig. 1c). Datlinger teaches the use of barcoded beads and the Drop-seq protocol, citing Macosko et. al. Macosko teaches the oligonucleotides attached to the beads are comprised of a primer to enable PCR amplification, a cell barcode, and a unique molecular identifier (Fig. 1).
Datlinger further teaches the cells were transfected (pg. 297, right col., par. 2-3; pg. 298, left col., par. 1, right col., par. 1; Online methods, PDF pg. 6, left col., par. 2-3, right col., par. 1; Online methods, PDF pg. 7, left col., par. 2-3, right col., par. 2-3). Datlinger teaches the cells stably express Cas9 after transduction with LentiCas9-Blast (Online methods: PDF pg. 6, left col., par. 3). Datlinger teaches the use of at least ten thousand cells (Fig. 1i; Online methods: PDF pg. 7, left col., par. 2; PDF pg. 7, right col., par. 3).
Datlinger teaches the application of Drop-seq after performing transduction of three cell lines (pg. 299, left col., par. 2), where reverse transcription was performed to generate cDNA (Online methods; PDF pg. 9, left col., middle of par. 1). Datlinger teaches the reverse transcribing is performed after breaking the partitions (Online methods; PDF pg. 9, left col., middle of par. 1).
Datlinger does not teach a mixture of cells and template particles being agitated to generate a plurality of partitions near simultaneously.
Meltzer teaches a method of providing capture template particles and an improved emulsion droplet-based target capture and barcoding method thereof (Abstract).
Meltzer teaches a plurality of partitions may be generated by agitating a mixture of target particles and template particles, wherein the template particles comprise attached polynucleotides (par. 0011). Meltzer teaches the template particles are tiny, generally spherical particles and may include a polymer such as a hydrogel (par. 0045). Meltzer further teaches the template particles may be microgel particles that are micron-scale spheres of gel matrix or a hydrogel (par. 0046-0047).
Meltzer teaches the method described provides an improved emulsion droplet-based target capture and barcoding in monodisperse droplets (par. 0034), and teaches the methods provide emulsions including droplets that are extremely uniform in size (par. 0081). Meltzer further teaches that microfluidic methods has disadvantages such as small inaccuracies in the fabrication of the devices can lead to device failure (par. 0084), and are non-ideal for generating double emulsions (par. 0085). Meltzer states that the present disclosure describes a flexible approach to capture and label targets of interest from biological samples, which leverages the particle-templated emulsification technique described in Hatori et al. and provides related advantages (par. 0002).
Hatori teaches the requirement of microfluidics to partition the sample into monodispersed droplets is a significant barrier that impedes implementation, and teaches that the ability to encapsulate samples in monodispersed droplets without microfluidics should facilitate the implementation of compartmentalized reactions in biology (Abstract). Hatori teaches this method is compatible with biological operations like cell culture and ddPCR, and should facilitate adoption of droplet compartmentalization for biological applications and advance the field of digital biology (pg. 9819, right col., par.2).
Regarding the combining, into a mixture, at least ten thousand of the cells with hydrogel particles comprising oligonucleotides and magnetic particles, Hatori further teaches that particle-templated emulsification is more scalable than conventional microfluidic emulsification because the time to emulsify a sample does not increase appreciably with sample volume, which makes it valuable for implementing droplet encapsulation into high throughput workflows (pg. 9819, left col., par.3). It would have been obvious to one of ordinary skill in the art to use a large number of cells as Datlinger teaches the use of at least ten thousand cells and Hatori teaches the particle-templated emulsification is more scalable than traditional microfluidic techniques; it therefore would have been obvious to one of ordinary skill in the art to use at least ten thousand cells in order to increase the amount of cells analyzed, with no evidence of unexpected results.
Datlinger, Meltzer, and Hatori do not teach magnetic particles wherein the magnetic particles are embedded within the hydrogel or the manipulation of the hydrogel template particles via the magnetic particles during the library preparation method.
Regev teaches methods for pooled screening of perturbations correlating to a phenotype, methods of in vivo perturbation screening, and high-throughput sample multiplexing (Abstract). Regev teaches that hydrogel particles can further comprise magnetic particles, which enable magnetic separation, aids in clean up and washing steps, and greatly enhances automation and throughput (par. 0535).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to substitute the method of partition generation taught by Datlinger with the method taught by Meltzer. The method taught by Meltzer was known to generate partitions encapsulating cells and template particles, and Meltzer teaches that this method allows for the creation of uniform droplets without the use of microfluidics. As Meltzer as evidenced by Hatori teaches, microfluidics have disadvantages such as inaccuracies that lead to device failure and allows for the implementation of droplet compartmentalization on a broader scale without the need for specialized hardware or skills (Hatori, pg. 9813, left col., par. 3).
Therefore, it would have been obvious to one of ordinary skill in the art to substitute the method of partition generation taught by Datlinger for the method taught by Meltzer, as Meltzer teaches this method allows for a wider adoption of droplet compartmentalization without the use of specialized hardware, with no evidence of unexpected results.
It would have further been obvious to one of ordinary skill in the art to include magnetic particles within the hydrogel, as Regev teaches the inclusion of magnetic particles within hydrogel particles, and provides motivation for the inclusion of magnetic particles as it enables magnetic separation, aids in clean up and washing steps, and greatly enhances automation and throughput (par. 0535), with no evidence of unexpected results. Furthermore, it would have been obvious to one of ordinary skill in the art to manipulate the hydrogel template particles via the magnetic particles during the library preparation method, as Regev teaches that the magnetic particles enable magnetic separation, aids in clean up and washing steps, and enhances automation and throughput.
Regarding claim 6, Datlinger teaches the CROP-seq lentiviral construct includes a gRNA cassette within the 3’ long terminal repeat, which is duplicated during viral integration. It expresses an RNA polymerase III transcript for genome editing and a polyadenylated RNA polymerase II transcript detected by single-cell RNA-seq (Fig. 1e). Datlinger further teaches the cells were transfected (Online methods; PDF pg. 6, right col., par. 3). Datlinger additionally teaches the gRNA becomes part of the mRNA transcribed by RNA polymerase II (pg. 297, right col., par. 2).
Regarding claim 9, Datlinger teaches a re-engineered LentiGuide-Puro construct was used to include the gRNA in a polyadenylated mRNA transcript (pg. 297, right col., par. 2). Datlinger additionally teaches the gRNA becomes part of the mRNA transcribed by RNA polymerase II, and functional gRNAs continue to be expressed (pg. 297, right col., par. 2).
Regarding claim 10, Meltzer teaches the agitation can be performed with a vortexer (par. 00105). It would have been obvious to one of ordinary skill in the art to agitate with a vortexer, as it would be a simple substitution of one method of agitation for another, with no evidence of unexpected results.
Regarding claims 11 and 17-18, Datlinger teaches the use of barcoded beads and the Drop-seq protocol, citing Macosko et. al. Macosko teaches the oligonucleotides attached to the beads are comprised of a primer to enable PCR amplification, a cell barcode, and a unique molecular identifier (Fig. 1).
Regarding claims 12 and 13, Datlinger teaches the cDNA is enriched using PCR (Online methods; PDF pg. 9, left col., middle of par. 1).
Regarding claims 14 and 21, Meltzer teaches that lysis of the target particles within the encapsulation may be performed by high temperature lysis reagent release from within the hydrogel matrix of the capture template particles (par. 0064). It would have been obvious to one of ordinary skill in the art to release lytic reagents contained inside the template particles using high temperatures, as it would be a simple substitution of one method of lysis for another, with no evidence of unexpected results.
Regarding claim 15, Meltzer teaches that the lysis buffer contains detergents (par. 00118). Meltzer does not specifically teach that the detergents are within internal compartments of the template particles. Meltzer does teach that lysis reagents can be released from within the hydrogel matrix of the template particles (par. 0064). It would be obvious to one of ordinary skill in the art that the lysis reagents within the hydrogel matrix includes detergents, as Meltzer teaches that lysing agents and buffers contain detergents (par. 00118), and it would be an obvious substitution with no evidence of unexpected results.
Regarding claims 19-20, Datlinger teaches the guide RNAs target certain genes (Online methods; PDF pg. 9, right col., par. 3). Datlinger does not teach the genes include an oncogene. Meltzer teaches that primers specific to genes of interest can be used to amplify the particular gene, such as oncogenes (par. 0074).
Therefore, it would have been obvious to target oncogenes using the method as taught by Datlinger and Meltzer, as Meltzer teaches that oncogenes are genes of interest, and it would be a simple substitution of one type of target gene for another, with no evidence of unexpected results.
Claim(s) 7-8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Datlinger, Meltzer and Regev as evidenced by Macosko and Hatori as applied to claim 1 above, and further in view of Delaney et. al. (US 2019/0233878 A1; previously cited).
Datlinger, Meltzer, Zhang, and Macosko do not teach the template particles comprise materials that provide the template particles with a positive surface charge to promote associating of cells with the template particles.
Delaney teaches a method for making a hydrogel bead in order to partition a sample, such a cell, for further processing (par. 0004, 0005). Delaney teaches the gel may be positively charged and comprise polyethyleneimine (par. 0253). Delaney further teaches this allows for a negatively charged component of a cell to interact with the positively charged component of the bead; one or more components from a cell may be capable of being retained due to the electrostatic interactions (par. 0253).
It would have been obvious to one of ordinary skill in the art to use materials such as polyethyleneimine to create a positive charge in the particles as taught by Delaney, Meltzer, Zhang, and Macosko in the method of claim 1, as Delaney teaches that this would allow for cell components to be retained within the bead, with no evidence of unexpected results.
Claim(s) 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Datlinger et al. in view of Meltzer et al. and Regev et al., and evidenced by Macosko et al. and Hatori et al. as applied to claim 1 above, and further in view of Shi et al. (Imparting functionality to the hydrogel by magnetic-field-induced nano-assembly and macro-response. ACS Applied Materials & Interfaces. 12, 2020, 5177-5194; previously cited).
Datlinger, Meltzer, and Regev do not teach that the magnetic particles are paramagnetic. Shi teaches that the inclusion of magnetic particles imparts functionality to hydrogels (Abstract). Shi teaches the inclusion of paramagnetic or superparamagnetic nanoparticles into the hydrogel, which results in the hydrogel exhibiting functionalities such as magnet-controlled release and magnetic separation (pg. 5184, right col., par. 3; pg. 5185, left col., par 1).
It would have been obvious to one of ordinary skill in the art to select a paramagnetic material as Shi teaches the inclusion of paramagnetic nanoparticles into hydrogel results in the hydrogel achieving functionalities such as magnet-controlled release and magnetic separation; furthermore, it would be a simple substitution of one type of magnetic particle for another, with no evidence of unexpected results.
Response to Arguments
Applicant's arguments filed 02/18/2026 have been fully considered but they are not persuasive.
Regarding the argument that Datlinger, Meltzer, Regev, Macosko, and Hatori fails to teach or disclose combining, into a mixture, at least ten thousand of the cells with hydrogel template particles comprising oligonucleotides and magnetic particles. This is not found persuasive. Datlinger teaches the use of over ten thousand cells (Fig. 1i; Online methods: PDF pg. 7, left col., par. 2; PDF pg. 7, right col., par. 3).
Furthermore, Hatori teaches that particle-templated emulsification is more scalable than conventional microfluidic emulsification because the time to emulsify a sample does not increase appreciably with sample volume, which makes it valuable for implementing droplet encapsulation into high throughput workflows (pg. 9819, left col., par.3). It would have been obvious to one of ordinary skill in the art to use a large number of cells as Datlinger teaches the use of at least ten thousand cells and Hatori teaches the particle-templated emulsification is more scalable than traditional microfluidic techniques; it therefore would have been obvious to one of ordinary skill in the art to combine, into a mixture, at least ten thousand cells with hydrogel template particles comprising oligonucleotides and magnetic particles in order to increase the amount of cells analyzed, with no evidence of unexpected results.
Regarding the argument that Datlinger does not teach or suggest one or more plasmids encoding a Cas endonuclease and guide RNAs for genome editing, this is not found persuasive. Datlinger teaches one or more plasmids encoding a Cas endonuclease and guide RNAs for genome editing throughout the Online Methods (Datlinger, pg. 6-10 of provided document). Furthermore, Datlinger teaches the cells were transfected (pg. 297, right col., par. 2-3; pg. 298, left col., par. 1, right col., par. 1; Online methods, PDF pg. 6, left col., par. 2-3, right col., par. 1; Online methods, PDF pg. 7, left col., par. 2-3, right col., par. 2-3). Datlinger teaches the cells stably express Cas9 after transduction with LentiCas9-Blast (Online methods: PDF pg. 6, left col., par. 3). Datlinger teaches the CROP-seq lentiviral construct includes a gRNA cassette within the 3’ long terminal repeat, which is duplicated during viral integration. It expresses an RNA polymerase III transcript for genome editing and a polyadenylated RNA polymerase II transcript detected by single-cell RNA-seq (Fig. 1e). Datlinger further teaches the cells were transfected (Online methods; PDF pg. 6, right col., par. 3). Datlinger additionally teaches the gRNA becomes part of the mRNA transcribed by RNA polymerase II (pg. 297, right col., par. 2).
Regarding the arguments presented in the remarks filed 12/11/2025, the response presented in the Advisory Action of 12/23/2025 is reproduced below:
Applicant argues that one of ordinary skill in the art would not be motivated to combine Datlinger, Meltzer, Regev, Hatori, and Macosko. Applicant argues that the alleged benefits associated with Regev's magnetic particles are described in the larger context of microfluidic devices, whereas Meltzer highlights an approach that is specifically microfluidics-free. Applicant states that when the disclosure of Regev is considered as a whole, the benefits of magnetic hydrogel particles are not taught independently of the microfluidic context in which they are embedded.
This is not found persuasive. Regev broadly teaches the benefits of magnetic particles, and does not teach that the magnetic hydrogel particles can only be used within microfluidic devices. Regev states that “magnetic particles enable magnetic separation, aiding in clean up and washing steps in multiple reactions. Not being bound by a theory, the use of magnetic particles greatly enhances automation and therefore throughput.” (par. 0475). One of ordinary skill in the art would be motivated by the general teaching of Regev that magnetic particles in hydrogels can enable magnetic separation and aid in clean up and washing steps to use magnetic particles in hydrogels for the benefits described by Regev. One of ordinary skill in the art would have a reasonable expectation of success as the benefits of the magnetic particles in hydrogel are described broadly, and one of ordinary skill in the art would expect that the magnetic particles in hydrogel would have similar properties when substituted into the method taught by Datlinger and Meltzer, with no evidence of unexpected results.
Therefore, Applicant’s arguments are not persuasive.
Conclusion
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/R.L.B./Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681
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