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 .
Application Status and Withdrawn Rejections
Applicant’s amendment filed March 30, 2026, amending claims 1, 8, 10, 14 and 18, and canceling claims 5-7 and 15-17 is acknowledged. Claims 1-4, 8-14 and 18-20 are pending. Claims 11-13 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected group, there being no allowable generic or linking claim. Claim 4 remains withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim.
Claims 1-3, 8-10, 14 and 18-20 are under examination.
In view of filing the foreign priority document (see next section), the §102 rejections over references Lin, Han and Zhang are withdrawn as none of the references are prior art under §102(a)(1). The amendment to claim 1 requiring the Cas13 to be dLwCas13a overcomes the §102 and §103 rejections over Hsu. The amendment to claim 10 overcomes the §112(b) rejection.
Claims 1 and 14 were amended to require nearly all the limitations of canceled claims 8 and 18, respectively. The §103 rejection over Zhang is maintained as it pertains to canceled claims 8 and 18 and new in regards to the dependent claims of claims 1 and 14, as necessitated by amendment.
Any other rejection or objection not reiterated herein has been overcome by amendment. Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Priority
Acknowledgment is made of applicant's claim for foreign priority based on a PCT application filed in China on February 25, 2020 – Application PCT/CN2020/076562. A certified copy of the PCT application was filed March 30, 2026. The effective filing date of the claimed invention is February 25, 2020.
Claim Rejections - 35 USC § 103 - Zhang
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, 8-9, 14, and 18-20 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (US 20190359971 A1, published November 28, 2019, priority to at least June 19, 2017; of record) as evidenced by Genbank (WP_021746774.1, https://www.ncbi.nlm.nih.gov/protein/WP_021746774.1, [retrieved 12/16/2025]; of record). This is a maintained rejection of claims 1 and 14 and a new rejection of all dependent claims necessitated by amendment to claims 1 and 14.
For logistical purposes, the rejections of claims directed to fusion proteins (14 and 18-20) are recited first followed by the rejections of claims directed to cells comprising the fusions proteins (1-2, 8-9).
Regarding claims 14 and 19, Zhang teaches “The ability of dC2c2 (dCas13a) to bind to specified sequences could be used… to… (iv) capture specific transcripts (… use of dC2c2 to localize biotin ligase activity [a proximity tagging enzyme] to transcripts) to enrich for proximal molecular partners including RNAs and proteins” (i.e., the Cas13a is catalytically dead and recruits biotin ligase to the targeted RNA) ([0015]). The Specification identifies LwaCas13a as WP_021746774.1 (Table B). Genbank teaches that accession WP_021746774.1 is Cas13a originating from Letotrichia wadei and was originally named C2c2. Thus, the dC2c2 taught in Zhang is inherently dLwaCas13a. Zhang teaches RNA-binding proteins can be identified using an RNA-targeting effector protein of the invention (i.e., dC2c2, dLwaCas13a) to label locally bound proteins with biotin ([0291]). Zhang also teaches dC2c2/dCas13a fused to a variety of effector proteins in order to localize the effector protein at targeted RNA ([0305], [0513]).
Zhang does not expressly teach that the biotin ligase is fused to dC2c2/dCas13a for recruitment.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have fused the biotin ligase to dLwCas13a. It would have amounted to the simple combination of known elements to yield predictable results. The skilled artisan would have predicted that the biotin ligase could actually be fused dLwCas13a because Zhang teaches many other effector domains fused to dLwCas13a for the purpose of recruiting the effector to the target RNA. The skilled artisan would have been motivated to do so because Zhang teaches protein fusions as a means for effector domain recruitment to a target RNA.
Regarding claim 18, Zhang teaches dLwaC2c2 was generated by making R474A and R1046A substitutions (i.e., arginine to alanine substitutions) in the two HEPN domains ([1028]).
Regarding claim 20, Zhang also teaches an NLS fused to catalytically inactive Cas13a that is also fused to an effector domain ([0126], [0047], Fig 45).
It would have also been obvious to one skilled in the art to have included an NLS fused to the dLwCas13a-biotin ligase fusion protein rendered obvious for claim 14. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have predicted that an NLS could be included because Zhang demonstrates an NLS included on other dCas13a-fusion proteins. One would have been motivated to do so for the purpose of labeling nuclear RNA-binding proteins that interact with the targeted nuclear RNAs.
Regarding claims 1-2 and 8, the teachings of Zhang regarding dLwCas13a and biotin ligase and the obviousness of fusing the two domains together are recited above as for claims 14 and 18-20 and incorporated here. Zhang teaches recombinant expression vectors comprising polynucleotides encoding the Cas13a proteins for expression in host cells ([0163]-[0164], [0740]). Zhang teaches expressing the Cas13a proteins in mammalian tissues, organs and organisms (i.e., a transgenic organism).
It would have been obvious to one skilled in the art to have introduced an expression vector encoding the obvious dLwCas13a-biotin ligase into a host cell and organism taught in Zhang. It would have amounted to the simple combination of known elements by known means to yield predictable results. The skilled artisan would have predicted that a dLwCas13a-biotin ligase could be encoded on a polynucleotide and introduced into cells of an organism because both dLwCas13a and biotin ligase are proteins and therefore can be genetically encoded. Additionally, Zhang teaches encoding dLwCas13a fusion proteins in polynucleotides and introducing the expression vectors into cells. The skilled artisan would have been motivated to do so in order to determine proteins that bind to RNAs normally in different cell types and under different conditions.
Regarding claim 9, Zhang teaches expression of Cas13a effectors can be modulated using inducible promoters that are known in the art ([0302], [0349], [0706]).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have expressed the obvious dLwCas13d-biotin ligase fusion from an inducible promoter in cells in an organism. It would have amounted to using known means to express an obvious fusion protein in cells to yield predictable results. The skilled artisan would have predicted the polynucleotides could be expressed using inducible promoters because Zhang teaches such genetic inducible systems are well-known in the art. One would have specifically been motivated to use an inducible promoter to prevent unwanted dLwCas13a-biotin ligase labeling in cells during transgenic cell selection.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang (US 20190359971 A1, published November 28, 2019, priority to at least June 19, 2017; of record), as applied to claims 1-2, 8-9, 14, and 18-20 above, and further in view of Liu (Liu et al., Nature Methods (2018), 15: 715-722). This is a new rejection necessitated by amendment to claim 1 and addresses the elected tagging enzyme PafA.
The teachings of Zhang are recited above and applied as for claims 1-2, 8-9, 14, and 18-20. Zhang also teaches proximity labeling technology employs an affinity tag to label polypeptides and RNAs in the vicinity of a protein or RNA of interest ([0278]). Zhang teaches the RNA targeting effector protein (i.e., dLwCas13a) can be used to target to label a molecule of interest ([0278]). Zhang teaches such technologies include Apex labeling ([0277]-[0278]) and can use biotin ligase ([0291]).
Zhang does not teach the proximity tagging enzyme is PafA.
Liu teaches a proximity-based tagging system called PUP-IT, in which a small protein tag called Pup (prokaryotic ubiquitin protein) is ligated onto nearby proteins by the PafA enzyme (Abstract; Fig 1). Liu teaches fusing PafA to a “bait” protein of interest to determine protein binding partners (Fig 1). Liu demonstrates fusion of PafA to CD28 (Fig 3), FRB (Fig 5) and IL-2 (Fig 6). Liu teaches developing their working model of the PUP-IT system with membrane proteins, but notes that applications of PUP-IT are not limited to membrane proteins (page 720, ¶2). Liu teaches the PUP-IT system is an alternative to the APEX and BioID system that label nearby substrates/proteins with biotin using a peroxidase enzyme or biotin ligase, respectively, fused to the protein of interest (page 715, ¶3 and page 720, ¶3). Liu teaches that PUP-IT appears to be more active than BioID in cells (page 720, ¶4). Liu describes the PUP-IT system as “complementary” to the APEX and BioID proximity labeling systems (page 720, ¶3).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have substituted the biotin ligase enzyme for Liu’s PafA enzyme in the dLwCas13a fusion protein rendered obvious for claim 1. It would have amounted to substituting one known proximity tagging enzyme for another by known means to yield predictable results. The skilled artisan would have predicted that PafA could be fused to dLwCas13a because 1) Zhang teaches a large variety of different domains fused to dLwCas13a and 2) Liu demonstrates PafA fused to at least three different cellular proteins. Because the prior art recognizes the equivalence of PafA and biotin ligase for the purpose of proximity tagging of proteins, an express suggestion to substitute one equivalent component or process for another is not necessary to render such substitution obvious. MPEP 2144.06.II. Nevertheless, the skilled artisan would have been motivated to make the substitution because Liu teaches that PafA is more active in cells than the biotin ligase of the BioID system.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang (US 20190359971 A1, published November 28, 2019, priority to at least June 19, 2017; of record), as applied to claims 1-2, 8-9, 14, and 18-20 above, and further in view of Buchman (US 20220145297 A1, priority to at least January 29, 2020). This is a new rejection necessitated by amendment to claims 1 and 10.
The teachings of Zhang are recited above and applied as for claims 1-2, 8-9, 14, and 18-20. As indicated above for claim 9, Zhang teaches expression of Cas13a effectors can be modulated using inducible promoters that are known in the art ([0302], [0349], [0706]).
Zhang does not teach the UAS element as part of an inducible promoter.
Buchman teaches Cas13 polypeptides operably linked to an inducible promoter ([0004]). Buchman teaches CasRx (i.e., a Cas13 polypeptide) linked to UAS elements within an inducible promoter for use of Cas13 in Drosophila models ([0014], [0076], [0098], FIGs 1, 14).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have expressed dLwCas13d-biotin ligase fusion from an inducible promoter comprising a UAS element. It would have amounted to using known means to express an obvious fusion protein in cells to yield predictable results. The skilled artisan would have predicted the obvious dLwCas13a fusion could be expressed using inducible promoters with UAS elements because Bachman demonstrates UAS-mediated inducible expression of a related Cas13 protein and Zhang teaches inducible systems are well-known in the art. One would have specifically been motivated to use a UAS-comprising inducible promoter for controllable expression in Drosophila systems as demonstrated by Bachman.
Response to Arguments - §103
Applicant argues that Zhang does not teach a fusion protein comprising dLwCas13a and a proximity tagging enzyme (Remarks, page 11, ¶1). This argument has been fully considered but is not persuasive because the rejection is one for obviousness, not anticipation. Therefore, the argument does not address the merits of the rejection.
Applicant argues that Zhang teaches C2c2, which is a generic Cas13a that constitutes a broad genus of proteins (Remarks, page 11, ¶1). This argument has been fully considered but is not persuasive because 1) Zhang teaches any of the Cas13a proteins can be fused to effector proteins, and 2) Zhang’s working examples are with the “C2c2” orthologues from Leptotrichia wadei, i.e., LwCas13a (see e.g., Zhang’s examples 3 and 7). Zhang even expressly teaches “dLw2C2c2” (i.e., dLwCas13a) fused to an effector protein ([0972]).
Applicant argues that in the field of RNA targeting using Cas enzymes, the performance of any particular species of Cas is highly unpredictable (Remarks, page 11, ¶1). This argument has been fully considered but is not persuasive. First Applicant does not provide evidence of this unpredictability. MPEP 716.01(c) makes clear that arguments of counsel cannot take the place of evidence in the record. Second, the working examples of targeting RNA in Zhang used LwCas13. Given that Zhang teaches targeting RNA using a dLwCas13a fusion protein, it would have been entirely predictable to make and use a different dLwCas13a fusion protein with any of the effectors listed in Zhang.
Applicant argues that the claimed invention, dLwCas13a fused to a proximity tagging enzyme such as PafA and TurboID/miniTurbo, has unexpected effects as it can effectively enrich RNA binding proteins and their associated distinct RNAs, which is not taught in the cited references (Remarks, page 11, ¶2). This argument has been fully considered but is not persuasive. First, except for claim 3 (and claim 4 which is not under examination), Applicant’s proffered evidence is not commensurate in scope with the claims. See MPEP 716.02(d). The claims are directed to dLwCas13a enzymes fused to a genus of proximity tagging enzymes including biotin ligase and ascorbate peroxidase. Second, unexpected results must be compared to the closest prior art. See MPEP 716.02(e). In this case the closest prior art is the Zhang (US 20190359971 A1, published November 28, 2019, priority to at least June 19, 2017) in view of Liu (Liu et al., Nature Methods (2018), 15: 715-722), whose teachings are recited in the §103 rejection of claim 3 above. Liu teaches the PafA proximity tagging technology is able to enrich proteins that interact with the tagged protein (Fig 4). Liu teaches that in the case of CD28, sixteen binding partners were enriched using the PafA technology (Fig 4). Therefore, Applicant’s proffered evidence of identifying four RNA-binding proteins that bind the RNA that dLwCas13a is targeted to is not unexpected or surprising.
Conclusion
No claims are 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635
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