DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office action is in response to the communication filed 10-23-25.
Claims 1, 3-7, 9-13 are pending in the instant application.
Response to Arguments and Amendments
Withdrawn Rejections
Any rejections not repeated in this Office action are hereby withdrawn.
Maintained Rejections
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 3-7, 9-13 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention. The teachings in the specification are not representative of the large genus of acceptor and donor oligonucleotides claimed for the reasons set forth in the Office action mailed 5-27-25 and as set forth below.
Applicant’s Arguments
Applicant argues the following:
…[T]he Office has alleged that the disclosure Figure 2, are not representative of the large genus of compounds claimed. Applicant respectfully disagrees for the following reasons. Figure 3 fully discloses all of the important claimed elements of both the donor and acceptor oligonucleotides that enable the claimed CRISPR Enabled DNA Synthesis method to function. Further, as pointed out by Examiner, the specification as originally filed amply points out the specification discloses: a 5’ donor oligonucleotide with a molecular beacon target site, a molecular beacon with a 5’ 6-FAM and 3’ Iowa Black, a synthetic oligonucleotide ligated to a cleaved product, a forward primer for amplifying a ligation product as shown in Figure 2, reverse primers used to amplify ligated products. Therefore, these teachings provide sufficient guidance to make the donor and acceptor oligonucleotides of the present invention and to practice the claimed invention.
Response to Applicant’s Arguments
Applicant's arguments filed 10-23-25 have been fully considered but they are not persuasive.
The breadth of the claims:
The claims are broadly drawn to donor oligonucleotides of any length comprising a partially double stranded sequence formed by a hairpin loop, at least a six nucleotide base overhang at the 5' end of the oligonucleotide, a blocked 3’ terminus, a sequence that is a protospacer adjacent motif (PAM), a sequence that is a RNA guided nuclease binding site, a nuclease cleavage site at least 1 base from the 5' terminus of the oligonucleotide, further comprising at the 5’ terminus at least one nucleotide, N, of a target DNA sequence, which oligonucleotide has a melting temperature greater than 65°C, or a plurality of oligonucleotides thereof, each with a unique 5’ terminus nucleotide or nucleotide subsequence, N, of a target DNA to be synthesized, which donor oligonucleotide is optionally complexed with a class II CRISPR/Cas Cpf1 nuclease and a gRNA at the protospacer adjacent motif (PAM) and nuclease binding site of the oligonucleotide, which oligonucleotide, guide RNA or nuclease are optionally modified with a purification tag optionally comprising biotin.
The claims are further drawn to acceptor oligonucleotides of any length comprising a partially double stranded sequence formed by a hairpin loop, at least a one nucleotide base overhang at the 3' terminus of the oligonucleotide, a PAM, a sequence that is a RNA guided nuclease binding site, a nuclease cleavage site at least one base from the 3' terminus of the oligonucleotide, and comprising at the 3’ terminus at least one nucleotide, N, of a target DNA sequence, which oligonucleotide is characterized by a melting temperature greater than 65°C, or a plurality of acceptor oligonucleotides each with a unique 3’ terminus nucleotide or nucleotide subsequence, N, of a target DNA, which oligonucleotide is optionally complexed with a class II CRISPR/Cas Cpf1 nuclease and a gRNA at the PAM and nuclease binding site of the oligonucleotide, which acceptor oligonucleotide, guide RNA or nuclease are modified with a purification tag optionally comprising biotin.
The claims are further drawn to methods of synthesizing a single stranded target DNA, the method comprising the steps of providing a plurality of donor and acceptor oligonucleotides of any lengths including donor oligonucleotides, donor oligonucleotides each with a unique nucleotide, or a subsequence of a target DNA sequence covalently bound to the 5’ terminus, acceptor oligonucleotides each with unique nucleotide, or subsequence of the target DNA sequence covalently bound to the 3’ terminus, determining a starting point and order of addition of nucleotides necessary to form a complete target single stranded DNA sequence, ligating the 5’ terminus of a donor oligonucleotide comprising N, a nucleotide or nucleotide subsequence determined to be the starting point, to the 3' terminus of an acceptor oligonucleotide to create a ligated product, contacting the ligated product with a guide RNA directed nuclease, to cleave the donor oligonucleotide leaving the N originating from the donor nucleotide covalently linked to the 3' terminus of the acceptor nucleotide, thus producing an extended acceptor oligonucleotide, purifying the extended acceptor oligonucleotide, contacting the extended acceptor oligonucleotide, containing N, with an additional donor oligonucleotide, and repeating ligating, cleaving and purifying steps repeatedly, extending the subsequence N with each cycle to obtain in the final step a complete single stranded target DNA.
Teachings in the specification
As stated previously, the specification teaches the following:
Example 1. Ligation [0053] Ligation of ssDNA (FIG 1A) can be accomplished with existing enzymes. In one aspect, the enzyme comprises a thermostable AppLigase, an ATP dependent enzyme requiring 5' pre-adenylated donors, which in the example case necessitated a two-step ligation, wherein donor oligonucleotides are first adenylated and then can be ligated to acceptor oligonucleotides with App Ligase. Mth RNA Ligase is used to convert phosphorylated 5' DNA to App (Adenylated) DNA.
Example 2. Cleavage of ssDNA at the 5' end of Donor oligonucleotides [0054] As can be seen in FIGIA, one of the key reactions in the CEDS process involves the gRNA targeted and Cpfl mediated cleavage of donor oligonucleotides leaving 5' nucleotides as an extension on acceptor oligos Cpfl, a class II CRISPR/Cas system can be used in this approach because it can cut 5' of its recognition sequence removing the predefined gRNA target sequence from the growing DNA. To evaluate the 5' donor cleavage step, we developed an assay reliant on a fluorescent molecular beacon as illustrated in FIG 2.
[0055] This beacon specifically binds to a donor oligonucleotide, and when bound fluoresces. When the donor oligonucleotide is cleaved, the beacon can no longer bind and preferentially forms a hairpin which quenches fluorescence, as a result a decrease in fluorescence indicates donor DNA cleavage. A synthetic donor oligonucleotide was cleaved with Cpfl nuclease, and then the detector (molecular beacon) was added.
[0056] Wild type Cpfl, as well as other CRISPR/Cas nucleases contain non-specific nuclease activity which is activated once initial gRNA cleavage occurs. This is of course an unwanted reaction which degrades the linear DNA to be synthesized. [0057] Referring specifically to FIG 2, Cpfl mediated cleavage during CEDS is demonstrated. (A) A donor oligonucleotide is mixed with a gRNA Cpfl complex, which first binds (1) and then cuts the oligo (ii). In step (iii), in the event the donor oligo is not cut, once the molecular beacon is added it can hybridize to the oligo resulting in fluorescence. In step (iv), in the event the donor oligo is cut, the molecular- beacon preferentially forms a hairpin quenching fluorescence. In (v), in the case of wild type Cpfl enzyme with non-specific nuclease activity, after binding and cleavage occurs, nuclease activity will degrade any ssDNA present including the molecular beacon, releasing fluorophore, and greatly increasing fluorescence. (B) Cleavage reactions were carried with or without heat treatment prior to the addition of the detector (molecular beacon). C) Results of cleavage assays and appropriate controls. Wild type or mutant Cpfl (as well as no enzyme controls) were premixed with gRNA and used to cleave a donor oligonucleotide. (D) Cut donors, were ligated to synthetic oligos, amplified by PCR, and cloned into plasmids prior to sequencing. (E) A sample chromatogram of Sanger sequencing of clones confirming the correct cutting and ligation position. Ligation should occur between the highlighted G and C Cutting successfully occurred 5' of the C..
[0058] Fortunately, a mutant Cpfl nuclease Cpfl* (Cpfl(Q1025G,El028G)) has been characterized, where non-specific nuclease activity has been abolished, enabling the CEDS process. As can be seen in Figure 2, the use of wild type Cpfl, leads to an increase in fluorescence when the beacon is added, this is due to non-specific cleavage of the beacon itself, eliminating any quenching. Heat treatment of the reaction to kill Cpfl activity before adding the beacon, eliminates the increased fluorescence. In contrast Cpfl*, has the expected decrease in fluorescence on the addition of the beacon consistent with cleavage of the donor oligonucleotide and a loss of non-specific nuclease activity. Cleaved donor oligonucleotides were successfully adenylated and ligated to an acceptor oligo amplified by PCR and cloned (FIG 2D), sequencing of these products (FIG2E) confirmed the correct cleavage and ligation position, and the success of cutting of the donor oligonucleotides.
Example 3: Cleavage of ssDNA at the 3' end of Acceptor oligonucleotides [0059] With the success of cutting the donor oligonucleotides we demonstrate the cleavage of the acceptor oligonucleotides. For the donor oligonucleotides, the disclosed method relies on cleavage of the non-target strand (NTS) 24 bp from the PAM site. However, the orientation of the target site on the acceptor oligo is such the target strand (TS) will instead be cleaved. TS cleavage occurs |9bp from the PAM site on the same strand that the gRNA binds to. As illustrated in Figure 3, we designed a hairpin at the 5' end of the acceptor oligonucleotide and create a double stranded PAM site. As shown, this assay will again use a molecular beacon to confirm cleavage (FIG 3A), followed by ligation and sequencing of the cleaved product (FIG 3B).
Example 4: gRNA binding to target DNA precludes molecular beacon binding 0060] Referring to FIG 5, gRNA binding to target DNA precludes molecular beacon binding in detail. In heat killed samples, the control, gRNA + Target, had the same low level of fluorescence as Cpfl*+gRNA+Target. This is due to the RNA binding to the target site and blocking the binding of the molecular beacon. To show this, RNAaseA was added and, as expected, the low level of fluorescence returned to uncut target levels.
Example 5: Automated Cycling and DNA synthesis [0061] An important requirement for CEDS is the ability to capture and release linear DNA fragments, in a high throughput and iterative fashion. This is needed to be able to build desired DNA sequences from individual fragments in parallel. Toward this goal, an automated CEDS process using a liquid handler is illustrated in FIG 4.
[0062] Referring specifically to FIG 4, automated CEDS is described. (A) A target DNA sequence, in this case an mCherry expression construct is first split into subsequences which are amenable to exponential synthesis, in this case, an 874 bp DNA fragment is broken into a 512 bp and smaller exponential subsequences from 256 bp to 2 bp. (B) Computationally. the sequence of each subsequence is then split until single nucleotides are reached. At this point all unique fragments (red pieces) and repeat sequences (gray) are identified, creating a minimal set of unique sequences of each size. (C). Starting with 4 unique donors (A,T,C, and G), iterative rounds of adenylation/ligation and cleavage are performed, using 384 well plates, temperature blocks and magnetic plates. After each ligation, the reaction can potentially be split into two fractions, one where the donor is cut leading to an extended acceptor, and one where the acceptor is cut, leading to an extended donor. Cpfl* which stays bound to the donor and or acceptor oligos as well as the gRNA are removed from the reaction via a biotin covalently attached to the gRNA and a pull down with magnetic streptavidin beads. Cleaned extended acceptors and donors are then rearrayed for the next rounds of ligations. After the final ligations are complete, both ends are cleaved, and the ssDNA product amplified by PCR.
[0063] To reiterate, a target DNA sequence is first divided into pieces which are amenable to exponential synthesis, next computationally, the sequences of each piece are split into half until single nucleotides are reached. At this point all unique fragments and repeat sequences are identified, creating a minimal set of unique sequences of each size. Starting with 4 unique donor oligos (A, T, C, and G), iterative rounds of adenylation/ligation and cutting are then performed, using 384 well plates, temperature blocks and magnetic plates for purification. After each ligation the reaction can potentially be split into two factions, one where the donor is cut leading to an extended acceptor, and one where the acceptor is cut, leading to an extended donor (FIG 4C). Cpfl* which stays bound to the donor and or acceptor oligos as well as gRNA are removed from the reaction via a biotin on the gRNA and a pull down with magnetic streptavidin beads. Cleaned extended acceptors and donors are then recombined for the next rounds of ligations. After the final ligations are complete, both ends are cleaved, and the ssDNA product amplified by PCR.
[
0064] The CEDS approach overcomes many of these challenges by enabling exponential single stranded DNA growth, for example 2 bp to 4 bp to 8bp to 16 bp, etc. This exponential growth enables DNA fragments of up to 10 kilobases in less than 14 cycles, reducing cycle number and compounding errors associated with oligo building technologies. In addition, as larger fragments are assembled as ssDNA and do not rely on hybridization of dsDNA for synthesis, we hypothesize that many issues currently limiting DNA synthesis methods such as secondary structures, and mis-hybridization will be minimized in the CEDs approach. Finally, the CEDS approach only requires a limited set of oligonucleotide sequences which can be purchased in bulk at high quality and reused for all synthesis projects, enabling large-scale multiplexed gene synthesis.
Cloning [0065] 6-His-MBP-TEV-FnCpfl was acquired from Addgene (Addgene ID 90094). Cpfl* was cloned via site directed mutagenesis using the oligos SEQ ID No:4 and SEQ ID NO: 5. T4 PNK …, T4 Ligase, and DpnI…were used in the KLD reaction. Expression and Purification of Cpfl and Cpfl* Expression and purification of Cpfl and Cpfl*is adapted from. Cpfl and Cpfl* genes were expressed from a pET vector with a N-terminal 6x his-tag, followed by an MBP tag and a TEV cleavage site. 500 ml of low salt LB with 100 ug/ml ampicillin were inoculated with Rosetta(DE3) cells… overnight culture containing each expression construct. The inoculated media was grown at 37°C until the OD600 reached 0.6 -1.0. A final concentration of 0.5 mM IPTG was added and the induction was allowed for 18 hours at 20°CThe culture was then harvested as 50 ml aliquots and frozen at -80°C until purification. The cell pellet was resuspended in 10 ml of Lysis Buffer (20 mM HEPES, pH 7.5, 0.5M KCI, 25 mM imidazole, 0.1 % Triton X-100) followed by 5 minutes of sonication (pulses with 10 sec on and 20 sec off) for cell disruption and the supernatant was applied to Ni2+ -NT A-agarose resin in a drop column. The column was tumbled at 4°C for 1 hour and then washed with 25 ml of Wash Buffer (20mM HEPES, pH 7.5, 0.3M KCl, 25 mM imidazole) and then eluted with 4 ml of elution buffer (20 mM HEPES, pH 7.5, 0.15M KCl, 250 mM imidazole). The elution was then concentrated and exchanged to 500 ul of TEV Reaction Buffer (50 mM Tris, pH 7.5, 0.5 mM EDTA, 1 mM DTT) using centrifugal filter …and supplemented with 200 units of TEV protease… The cleavage was allowed at 4 °C for 72 hours. The reaction was then applied to Ni 7*-NTA-agarose resin to remove TEV protease and exchange to Storage Buffer (20 mM Tris, 0.15 M NaCl, 25% Glycerol) and stored at -20°C until use.
Single-stranded DNA Cleavage assay [0066] Cleavage assays were performed using purified Cpfl or Cpfl*. 350nM of Cpfl was used along with 700nM of crRNA and 35nM of 5' Donor Oligonucleotide. Buffer 3.1 (NEB #7203S) was supplemented with 5mM DTT Total reaction volume was 10uL. First, Cpfl was pre-incubated with crRNA for10 min at room temperature. 5' Donor Oligonucleotide was added, and the reaction was incubated at 37°C for 15 min. Samples were then either left on ice or denatured at 95°C for 10 min. To prevent RNA annealing to uncut ssDNA at the target site (FIG. 5), RNase A (GoldBio Cat# R-050-1) was added to the heat killed samples (final concentration of 100 ng/mL) while an equal volume of water was added to the non-heat treated samples. Samples were then incubated with the molecular beacon (SEQ ID NO: 15) for 10 min at room temperature and fluorescence was measured with excitation and emission at 492nm and 535nm, respectively.
Adenylation [0067] Adenylation was carried out using Mth RNA Ligase (NEB #E261 OS). The reaction was carried out by adding 10 uL of the heat killed Cpfl* reaction to the manufacturer's recommended protocol: 2 uwL of Mth RNA Ligase, 2 uL of 10X 5 DNA
Adenylation Reaction Buffer, 2 wL of 1mM ATP, and 4 uL of water for a total reaction volume of 20 wL. The reaction was incubated at 65°C for 1 hour and then heat killed at 85°C for 5 minutes
Ligation Assay [0068] Ligations were carried out using Thermostable 5' App RNA/DNA Ligase…. The adenylated Cpfl* reaction was ligated with an oligonucleotide (SEQ ID NO: 14) as described in Figure 2. The 20 uL ligation reaction was carried out with 14 uL of adenylated Cpfl*, 1.2 uL of 5 uM SEQ ID NO: 14, 2 uL of NEBuffer 1, 2 uL of 50 mM MnC 12, and 2 uL of Thermostable 5' App RNA/DNA Ligase. The reaction was incubated at 65°C overnight and then heat killed at 95°C for 5 minutes. The ligated product was then PCR amplified with SEQ ID NO: 17 and SEQ ID NO:18 using Econotaq DNA Polymerase (Lucigen #30035-1). The PCR product was purified and cloned via Golden Gate assembly using T4 DNA Ligase… and Esp3i… into SEQ ID NO: 19. Five clones were sent for Sanger … with sequencing primer SEQ ID NO: 20.
Contrary to Applicant’s assertions, the teachings in the specification, including the procedures described above, comprising primers to make Cpt1* from Cpf1, a 5’ donor oligonucleotide with a molecular beacon target site, a molecular beacon with a 5’ 6-FAM and 3’ Iowa Black, a synthetic oligonucleotide ligated to a cleaved product, a forward primer for amplifying a ligation product as shown in Figure 2, reverse primers used to amplify ligated products in Figure 2, and a plasmid used for Golden Gate assembly with a PCT of the ligated product in Figure 2, are not representative of the large genus of compounds claimed. The acceptor and donor oligonucleotides and their particular components or subcomponents, including the lengths of the partially double stranded sequences formed by a hairpin loop, the concise placement and sizes of the protospacer adjacent motifs, of the RNA guided nuclease binding sites, and the concise placement of the nuclease cleavage sites in the donor or acceptor oligonucleotides are not adequately described in the claims or in the specification.
Thus, Applicant was not in possession of the broadly claimed genus of modified donor or acceptor oligonucleotides.. For these reasons, the instant rejection is properly maintained.
Conclusion
THIS ACTION IS MADE FINAL. 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.
Certain papers related to this application may be submitted to Art Unit 1637 by facsimile transmission. The faxing of such papers must conform with the notices published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 C.F.R. ' 1.6(d)). The official fax telephone number for the Group is 571-273-8300. NOTE: If Applicant does submit a paper by fax, the original signed copy should be retained by applicant or applicant's representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED so as to avoid the processing of duplicate papers in the Office.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jane Zara whose telephone number is (571) 272-0765. The examiner’s office hours are generally Monday-Friday, 10:30am - 7pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Jennifer Dunston, can be reached on (571)-272-2916. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (703) 308-0196.
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Jane Zara
11-25-25
/JANE J ZARA/Primary Examiner, Art Unit 1637