DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 05/14/2026 has been entered.
Claim Status
Claims 16-20, 22-23, 40, 63-67 and 69 are pending.
Claims 63-67 and 69 are withdrawn from examination as being part of non-elected groups of inventions.
Claims 16-20, 22-23 and 40 are being examined.
All previous objections and rejections not set forth below have been withdrawn in view of applicant’s amendments to the claims.
Claim Rejections - 35 USC § 103
Claims 16-18, 20, 23 and 40 are rejected under 35 U.S.C. 103 as being unpatentable over Ye et al. (Programmable DNA repair with CRISPRa/I enhanced homology-directed repair efficiency with a single Cas9, 2018, Cell Discovery, 4:46) in view of Selma et al. (Strong gene activation in plants with genome-wide specificity using a new orthogonal CRISPR/Cas9-based programmable transcriptional activator, 2019, Plant Biotechnology Journal, 17:1703–1705) and Lowder et al. (Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems, 2017, in Kerstin Kaufmann and Bernd Mueller-Roeber (eds.), Plant Gene Regulatory Networks: Methods and Protocols, Methods in Molecular Biology, vol. 1629, DOI 10.1007/978-1-4939-7125) and in evidence of Kumaran et al. (Gene technologies in weed management: a technical feasibility analysis, 2020, Current Opinion in Insect Science, 38:6–14)
Claim 16 is drawn to a system for activating expression of a target nucleic acid, the system comprising:
a nuclease active Cas polypeptide, or a polynucleotide encoding the nuclease active Cas polypeptide;
a first dead guide polynucleotide comprising a 14 to 16 nucleotide spacer sequence that mediates increased expression of the first target nucleic acid, wherein the first dead guide polynucleotide comprises an aptamer;
a polypeptide comprising an adapter domain and a multimerized epitope, wherein the adapter domain binds the aptamer, or a polynucleotide encoding the polypeptide;
a polypeptide comprising an affinity domain and a transcriptional activation domain, wherein the affinity domain binds the multimerized epitope, and wherein the transcriptional activation domain is two repeats of a TAL activation domain (2xTAD), or a polynucleotide encoding the polypeptide; and
a second dead guide polynucleotide comprising a 14 to 16 nucleotide spacer sequence that mediates reduced expression of the second target nucleic acid and/or a guide polynucleotide that mediates sequence-specific cleavage at a target site in the genome.
Ye et al. describes an effective, flexible, safer strategy to perform both CRISPRa/i (“a” refers to activation and “i” refers to interference or repression) and precise gene editing mediated by catalytically dead guide RNAs (dgRNAs) and a catalytically active Cas9 (abstract) (as recited in claim 17). During the gene editing process, Cas9-sgRNA complex is known to induce double stranded breaks (DSBs) which is then repaired by either non-homologous DNA end joining (NHEJ) or homology directed repair (HDR) (page 2, left column, para 2, line 7). The error prone repairs via NHEJ pathway often introduce unpredictable indel mutations in the target gene(s) (page 2, left column, para 2, line 7-9). NHEJ has been considered the major pathway to repair the DNA in mammals (page 1, right column, para 1, line 10-11) and plants (Kumaran et al.; page 10, left column, para 3, line 4-5). NHEJ is known to be more error prone in plants than any other organism (Kumaran et al.; page 10, left column, para 3, line 5-6). One of ordinary skill in the prior art therefore understands that HDR is more desirable to reduce or eliminate unpredictable indel mutations besides minimizing off-target gene editing, gene activation, and/or gene repression/interference.
Ye et al. describes catalytically dead guide RNAs (dRNA or dgRNA) of 14-15 nucleotide long retaining its binding ability to a target DNA sequence and allow catalytically or nuclease active Cas9 polypeptide (as recited in claim 17) to modulate gene expression (page 2, left column, para 2, line 23-25) i.e., undertaking CRISPR activation (CRISPRa) and interference (CRISPRi). The 14-15 nucleotide long dgRNA reads on to “comprising a 14 to 16 nucleotide long spacer sequence”, as recited in instant claim 16. Ye et al. also describes two different dead guide RNAs (dgRNAs or dRNAs) by- i) the “first dead guide polynucleotide” sequence is made by fusing the dgRNA scaffold sequence to Com binding loop (aptamer) to repress the NHEJ-related gene(s) like KU80 (Fig. 3a-b), and ii) the “second dead guide polynucleotide” sequence is made by fusing the dgRNA scaffold sequence to MS2 binding loop (aptamer) for recruiting (MS2 binding protein) MCP (as recited in claim 18) to activate HDR-related gene(s) (page 2, right column, para 1, line 1-5) like CDK1 (Fig. 3a-b). Transcription activation of CDK1 gene is achieved by P65 transcription activation domain (as part of the MCP-P65-HSF1 fusion protein) while transcriptional repression is achieved by KRAB (transcription repression) domain (as part of the COM-KRAB fusion protein (page 2, right column, para 1, line 1-5). Ye et al. also describes many expression vectors (as recited in claim 23) comprising various components to activate HDR pathway genes (p.4, Fig. 1).
However, Ye et al. does not describe a multimerized epitope or Transcription Activator-Like (TAL) activation domain (TAD).
Selma et al. describes strong gene activation in plants using CRISPR/Cas9 based system (title and abstract). Selma et al. teaches transcriptional activators based on CRISPR/Cas9 architecture, which combine several autonomous transcriptional activation domains (TADs) (also known as Transcription Activator-Like, TAL; and Transcription Activator-Like Effector Transcription Factors, TALE-TFs) (reads on to two repeats of TAD, 2xTAD) capable of recruiting the cellular transcription machinery, with the easily customizable DNA-binding activity of nuclease-inactivated (or catalytically inactive) Cas9 protein (p. 1703, left column, para 1, line 5-11 and line 13-22). The SunTag strategy uses multiepitope tags to attach multiple TADs to a single dCas9 protein resulting in higher activation rate (p. 1703, left column, para 1, line 14-19).
Selma et al. teaches the MS2 aptamer (page 1703, right column, line 11) and the adapter domain (MS2 viral Coat Protein, MCP) that binds to the MS2 aptamer (page 1703, right column, lines 10-11); a polypeptide comprising multimerized or multiepitope tags (viz. GCN4), as recited in claims 16 and 19 (page 1703, left column, para 1, line 18) such as GCN4, which is attached to an affinity domain (ScFv) (as recited in claim 20) (page 1703, right column, para 1, lines 8-9); and transcriptional activation domains like VP64 fused to the GCN4-ScFv fusion polypeptide (page 1703, right column, line 8-9).
Lowder et al. describes multiplexing transcriptional activation including via Transcription Activator-Like Effector Transcription Factors, TALE-TFs (p.167, para 1, line 11-12) or repression using CRISPR-dCas9-based systems in a plant (as recited in claim 40) (title and abstract) by expressing different gRNAs simultaneously (page 169, last para, last 2 lines). It teaches simultaneous activation and repression of two different genes using different guide RNAs (gRNAs) (Abstract). Lowder et al. describes two different sets of gRNAs to target two different genes (AtPAP1 and AtCSTF64) in the same genome in Arabidopsis (page 167, Abstract). The first group of gRNAs are used to activate AtPAP1 gene (page 172, Table 1) while the second group of gRNAs are used to repress AtCSTF64 gene (page 172, Table 2). Lowder et al. teaches transcriptional activation by using the transcriptional activation domain (VP64) while achieving transcriptional repression by using the transcriptional repression domain (SRDX) (page 167, abstract, line 4-6; page 168, para 3, line 1-5; page 169, Fig. 1).
Before the effective filing date of this application, it would have been obvious to one of ordinary skill in the art to develop a system of simultaneous transcriptional activation and repression of different but specific genes by using two different sets of dead guide RNAs (dgRNAs), as described by Ye et al. and Lowder et al. One set of the dgRNA(s) are attached to at least one activation domain and the second set of dgRNA(s) are attached to at least one repressor domain, as taught by Lowder et al. The specificity of the target gene(s) would have been decided by the dgRNA sequence.
Before the effective filing date of this application, an ordinarily skilled artesian would have been motivated to develop a system by using Cas9 polypeptide based technique that includes: two sets of dead guide RNAs each comprising aptamer(s), adapter domain binding specific aptamer sequence, multimerized epitope, an affinity domain, and a transcription effector which includes an activator or a repressor, as discussed above. The nucleotide-protein complex would have the ability to simultaneously active and repress transcription of two different set of target genes, and, thus, would regulate in-vivo gene expression resulting in higher activation/repression rate, as described by Selma et al.
Regarding claim 23, Ye et al (Fig. 1) and Lowder et al. (page 171, Fig. 2) describe several vectors containing different components of the system. It used specific vector for different steps of the method (i.e., the system of instant claim 16 wherein components are located on one or more vectors).
Regarding claim 40, Selma et al. uses tobacco plant (Nicotiana benthamiana) (page 1703, left column, para 2, line 2-3) to activate gene using the CRISPR-Cas based transcriptional activation system, as described before (i.e., a plant comprising the system of instant claim 16).
Claim 19 are rejected under 35 U.S.C. 103 as being unpatentable over Ye et al. in view of Selma et al. and Lowder et al., as applied to claims 16-18, 23 and 40 above, and further in view of Jacobsen et al. (US 20200017869A1).
Claim 19 depends from claim 16, wherein the multimerized epitope comprises 10 copies of a GCN4 epitope. Claim 20 also depends from claim 16, wherein the affinity domain comprises scFv.
Ye et al. in view of Selma et al. and Lowder et al. describes a nuclease active Cas9 polypeptide based system of transcriptional activation and repression by using two different sets dead guide RNAs that contain an aptamer (MS2), an adapter domain polypeptide (MCP) that binds to the (MS2) aptamer at one end and is fused to a multimerized epitope (GCN4) at the other end. The multimerized epitope binds to an affinity domain (ScFv), which, in turn, binds to either a transcriptional activation domain (VP64) or a transcriptional repression domain (SRDX) to simultaneously activate specific gene(s) while repressing different gene(s) as directed by two different sets of dgRNAs, as described above. Selma et al. describes the ScFv affinity domain attached to the GCN4 epitope (page 1703, right column, para 1, line 9-10) as recited in claim 19.
However, Ye et al. in view of Selma et al. and Lowder et al do not specify the number (10) of GCN4 epitopes used, as recited in claim 19.
Jacobsen et al. (US 20200017869A1) describes using at least 2 to 24 copies of the GCN4 epitope (page 11, para 0127). Jacobsen et al. also describes specifically using 10 copies of the GCN4 epitope (page 27, para 0323, line 4, 10; para 0324, line 3, 9). Using any specific number of GCN4 epitopes in a multimerized form is within the design choice of a person having ordinary skill in the art without affecting the outcome.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Ye et al. in view of Selma et al. and Lowder et al., as applied to claims 16-18, 23 and 40 above, and further in view of Brezgin et al. (Dead Cas Systems: Types, Principles, and Applications, 2019, Int. J. Mol. Sci., 20:6041).
Claim 22 depends from claim 16, wherein the Cas polypeptide is fused to a deaminase domain.
Ye et al. in view of Selma et al. and Lowder et al. describes a nuclease active Cas9 polypeptide based system of transcriptional activation and repression by using two different sets dead guide RNAs (dgRNAs) that contain an aptamer (MS2), an adapter domain polypeptide (MCP) that binds to the (MS2) aptamer at one end and is fused to a multimerized epitope at the other end. The multimerized epitope binds to an affinity domain (ScFv), which, in turn, binds to either a transcriptional activation domain or a transcriptional repression domain to simultaneously activate specific gene(s) while repressing different gene(s) as directed by two different sets of gRNAs, as described above.
However, Ye et al. in view of Selma et al. and Lowder et al. do not describe any deaminase domain.
Brezgin et al. describes systems comprising a Cas protein fused to cytidine or adenosine deaminases (page 12, para 5, line 1). The use of adenosine deaminases or adenine base editors effectively convert A-T base pairs to G-C base pairs in DNA (page 12, para 5, line 6-10). It also describes that specific Cas polypeptide (e.g., Cas type VI) directly interacts with target RNA molecules independently of PAM sequence and, thus, allowing the Cas-bound deaminase to edit RNA molecules (page 13, para 3, line 1-2). Compared to DNA editing, RNA editing has several important advantages, including a wider range of potential sites and direct RNA editing by deaminases without the assistance of endogenous repair systems (page 13, para 5, line 1-3). The RNA based gene editing system is more specific and does not exhibit significant off-target binding or RNA editing (page 13, para 4, line 5-7).
Before the effective filing date, it would have been obvious to one of ordinary skill in the art to further modify the system described by Ye et al. in view of Selma et al. and Lowder et al. by replacing the transcription activation/repression domain with deaminase protein or a deaminase domain of a deaminase protein as described by Brezgin et al. Fusing the deaminase protein or a deaminase domain of a deaminase protein with a nuclease active Cas9 polypeptide (as described by Ye et al.) and using dead guide RNAs would have enabled an ordinarily skilled artisan to perform targeted mutagenesis by converting A-T base pairs to G-C base pairs in DNA or in RNA sequences, as taught by Brezgin et al.
Before the effective filing date, an ordinary skilled artisan would be motivated to fuse the nuclease active Cas polypeptide with a deaminase domain while using dead guide RNAs with the realistic goal to introduce specific substitution mutation(s) by converting specific A-T base pairs to G-C base pairs in a DNA or in a RNA sequence, and reducing off-target mutation(s) while editing RNA sequences directly. Besides DNA editing (as described in this invention and by Ye et al., Selma et al., and Lowder et al.), RNA editing has its own advantages as it is more specific and does not exhibit significant off-target binding or RNA editing, as taught by Brezgin et al.
Response to Applicant’s Arguments
The argument set forth in the Applicant’s replies on 3/18/2026 has been fully considered but is not found persuasive.
Applicant argues, "Neither Ye, Selma, nor Lowder teaches or suggests use of a TAL activation domain" (response, p.6, para 1, line 1). The Applicant claims of "unexpected results" (page 6, para 4, line 2-3) based on the assumption of “two new activators, 2xTAD and 2xTAD-VP64, were developed and compared with the previously reported activators VP64, TV, and VPR. The systems with the 2xTAD activator showed the highest gene activation, about 250-fold when used in combination with the 10xGCN4 SunTag” with the reference to Fig. 3C (bridging paragraph between p.6-7).
The Examiner disagrees. Use of TAL domain (also known as TAL effector, TALE, targeting domains) is well known and well characterized in the art as also described by Jacobsen et al. (page 10 para 2). Selma et al. describes autonomous transcriptional activation domains (TADs) (also known as Transcription Activator-Like, TAL; and Transcription Activator-Like Effector Transcription Factors, TALE-TFs) capable of recruiting the cellular transcription machinery (p. 1703, left column, para 1, line 5-11 and line 13-22). Lowder et al. teaches transcriptional activation including via Transcription Activator-Like Effector Transcription Factors, TALE-TFs (p.167, para 1, line 11-12). Jacobsen et al. describes many DNA binding domains including TAL (Transcription Activator Like) effector targeting domains (p.10, para 0112, line 4-6). Jacobsen et al. also teaches that that methods of modifying TAL activation domains are well known in the art (page 10, para 0113, line 9-10).
The Examiner does not agree that the results presented here in the invention and/or as claimed is actually surprising and not obvious, as discussed in detail above. In short, 2xTAD is not any new development and was known in the prior art, including the art discussed above. Selma et al. describes combining several autonomous transcriptional activation domains (TADs) (reads on to two repeats of TAD, 2xTAD) capable of recruiting the cellular transcription machinery, with the easily customizable DNA-binding activity of nuclease-inactivated (or catalytically inactive) Cas9 protein (p. 1703, left column, para 1, line 5-11 and line 13-22). Selma et al. also describes VP64 (page 1703, right column, line 8-9) and the SunTag strategy which uses multiepitope tags to attach multiple TADs to a single dCas9 protein resulting in higher activation (p. 1703, left column, para 1, line 14-19).
Moreover, use of any specific effector protein including TAD/TALE for targeted gene activation is highly dependent on both the specific gene(s) being targeted, especially the specific sequence in its regulatory region(s) where transcription activation/repression takes place, and/or the host organism where it is being used (Moore et al. Transcription Activator-like Effectors: A Toolkit for Synthetic Biology, 2014, ACS Synth. Biol., 3:708-716; p. 713, left column, para 1, line 1-5; last para, last 8 lines). Thus, use of any specific combination of effector(s) including TAD as described in the invention and as claimed might give very different result in other plant species and/or other set of target gene(s). The use of 2xTAD and the results as described in the invention are not surprising. Further note that the activator in Fig. 3C that provided the 250-fold activation is 2xTAD when used in combination with 10xGCN4 SunTag, which is not required by independent claim 16. Applicant’s allegation of unexpected results is unpersuasive also because it is not commensurate in scope with the claims herein.
Conclusion
No claim is allowed.
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J.C.
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
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