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 .
Claim Status
Claims 1-2, 5, 11-12, 14-19, 22-24, 26-27, 31-32, 34, 36, 39, 41, 43-44, and 46-47 are pending.
Claims 1-2, 5, 11-12, 14-19, 22-24, 26-27, 31-32, 34, 36, 39, 41, 43-44, and 46-47 are examined on the merits.
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.
Claims 1, 11-12, 14-19, 22-23, 31-32, 39, 43, and 46-47 are rejected under 35 U.S.C. §103 as being unpatentable over Yang (Lei Yang et. al., Nature Biotechnology (01/02/2023) volume 41, pp958–967), in view of Fuser (Friedrich Fuser et. al., The Plant Journal (2014) 79, 348–359).
Claim 1 recites the method of editing a genomic target in a scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a Cas9 nicks or Cas12 nuclease and a guide RNA for the Cas9 nicks or Cas12 nuclease, wherein the nucleic acids are fused to nucleic acid encoding a meristem transport segment (MTS), wherein the genome of a cell in a meristem of the scion is edited.
Yang teaches a method of producing heritable transgene-free genome edits by grafting wild-type shoots/scions to transgenic donor rootstocks expressing mobile CRISPR reagents (Abstract). Yang further teaches designing fusions of Cas9 and guide RNA transcripts to tRNA-like sequence motifs (TLS) that move RNAs from transgenic rootstocks to grafted wild-type shoots/scions and achieve heritable gene editing in Arabidopsis thaliana and Brassica rape (Abstract; p958, left column). Yang further taches that Cas9-TLS and gRNA-TLS transcripts move from rootstock to scion, are detected in scion tissues including leaves, stems, flowers, and siliques, and induce genome edits in grafted wild-type scion tissues and progeny seed (p961, left column, pa2; p963, right column, pa1).
Yang further teaches that the mobile Cas9-TLS and gRNA-TLS fusion transcripts could be transported to meristems, thereby giving rise to a genome-edited lineage and edited germline progenitor cells producing the next generation (p961, left column, pa2).
Yang does not expressly teach that the Cas nuclease is a Cas9 nicks.
Fuser teaches that Cas9 can be engineered to function as a nicks by introducing an inactivating point mutation in one nuclease domain, including a Cas9-D10A nicks (Abstract; p349, right column, pa2). Fuser further teaches use of CRISPR/Case-based nicks for genome engineering in Arabidopsis thaliana (Abstract; p349, right column, pa3).
It would have been obvious to a person of ordinary skill in the art to substitute the Cas9 nuclease of Yang with the Cas9 nicks of Fuser, because Yang teaches a graft-mobile CRISPR delivery platform using Cas9/gRNA-TLS transcripts, and Fuser teaches that Cas9 nicks were known Cas9-based genome engineering reagents in plants. A person of ordinary skill in the art would have had reason to use the known Cas9 nicks in Yang’s graft-mobile editing systems as an alternative Cas9-based editing reagent, including to induce single- strand breaks, promote homologous recombination, or reduce undesired double-strand-break effects.
Accordingly, claim 1 is obvious over Yang and Fuser.
Claim 12 recites the method of claim 1, wherein the nucleic acid encoding the Cas9 nicks or Cas12 nuclease and the nucleic acid encoding the guide RNA are provided to the rootstock in different vectors.
For the same reason set forth above with respect to claim 1, Yang teaches Cas9 and gRNA constructs and transgenic lines expressing Cas9 and gRNAs, including lines in which Cas9-expressing plants and gRNA-expressing plants were crossed to combine the components (Supplementary Table 4, “Binary Cas9 and gNIA1 Plasmids Created in the Study”; p692, both left and right column).
Accordingly, claim 12 is primer facie obvious over Yang and Fuser.
Claim 14 recites the method of claim 1, wherein the scion and the rootstock are different plant species. Claim 15 recites the method of claim1, wherein the scion and the rootstock are the same plant species.
For the same reason set forth above with respect to claim 1, Yang teaches heterografting Brassica rape scions onto Arabidopsis transgenic root stocks expressing mobile Cas9-TLS and gRNA-TLS constructs (p963, right column pa1). Yang further teaches homografting Arabidopsis wild-type scions onto Arabidopsis transgenic rootstocks expressing mobile Cas9-TLS and gRNA-TLS constructs (p963, left column pa1), thereby teaching that the scion and rootstock may be different plant species or may be the same plant species.
Accordingly, claims 14-15 are primer facie obvious over Yang and Fuser.
Claim 16 recites the method claim 1, wherein the scion and/or rootstock is a dicot. Claim 17 recites the method claim 1, wherein the scion and/or rootstock is a monocot. Claim 18 recites the method of claim 1, wherein the scion is soy, canola, alfalfa, corn, oat, sorghum, sugarcane, banana or wheat.
For the same reason set forth above with respect to claim 1, Yang teaches Arabidopsis thaliana and Brassica rape and editing, Arabidopsis thaliana and Brassica rape are dicot plants. Yang further teaches grafting in monocot species (p966, left column, pa3). Yang teaches A. thaliana and Nicotiana sp. can easily be used as rootstocks for a very wide range of quite distantly related species including tomato, carrots, soybean, and onions (p966, left column, pa2).
Accordingly, claims 16-18 are primer facie obvious over Yang and Fuser.
Claim 19 recites the method of claim 1, wherein the MTS comprises: i. a meristem transport component (MTC); ii. an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop; iii. a Flowering Locus T (FT-) derived sequence, wherein the FT-derived sequence comprises the nucleotide sequence set forth in SEQ ID NO: 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19,20,21,22,23, or24; or iv. a tRNA-like sequence (TLS), wherein the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.
For the same reason set forth above with respect to claim 1, Yang teaches a tRNA-like sequence (TLS1) fused to the guide RNA to confer long-distance RNA mobility across the graft junction (p958, left and right column). Sequence alignment demonstrates that Yang’s TLS1 contains a fragment that is 100% identical to the nucleotide sequence of SEQ ID NO: 29 (see below). Although Yang’s TLS1 includes three additional nucleotide at 3’end, the claimed MST “comprises” sequence ID NO: 29. Therefore, Yang teaches a meristem transport segment comprising the claimed sequence.
Accordingly, claim 19 is primer facie obvious over Yang and Fuser.
PNG
media_image1.png
324
975
media_image1.png
Greyscale
Claim 22 recites the method of claim 1, wherein the nucleic acid encoding the MTS is located 3' of the nucleic acid encoding the Cas9 nicks or Cas12 nuclease and/or 3' of the nucleic acid encoding the guide RNA. Claim 23 recites the method of claim 1, wherein the nucleic acid encoding the MTS is located 5' of the nucleic acid encoding the Cas9 nicks or Cas12 nuclease and/or 5' of the nucleic acid encoding the guide RNA.
For the same reason set forth above with respect to claim 1, Yang teaches Cas9-TLS1/TLS2 and gRNA-TLS1/TLS2 constructs (Supplementary Data 1, a (2 and 3); b (2 and 3)), the tRNA-like sequence mobility motif (TLS1 or TLS2) is fused downstream of the Cas9 coding sequence and downstream of the guide RNA sequence. Thus, Yang teaches that the nucleic acid encoding the MTS located 3’end of the nucleic acid encoding the Cas9 editing reagent and/or 3’end of the nucleic acid encoding the guide RNA.
Yang further teaches gRNA-TLS1/TLS2 constructs (Supplementary Data 1, b(2 and 3)), the tRNA-like sequence mobility motif (TLS1 or TLS2) is fused upstream of the guide RNA sequence. Thus, Yang teaches that the nucleic acid encoding the MTS located 5’end of the nucleic acid encoding the guide RNA.
Accordingly, claims 22 and 23 are primer facie obvious over Yang and Fuser.
Claim 31 recites the method of claim 1, wherein the nucleic acid encoding the guide RNA is operably linked to a promoter. Claim 32 recites the method of claim 31, wherein the promoter is an RNA polymerase II promoter or an RNA polymerase III promoter.
For the same reason set forth above with respect to claim 1, Yang teaches that the guide RNAs are driven by U6-26 and U6-29 promoter, which are RNA polymerase III promoter (p958, right column, pa2).
Accordingly, claims 31-32 are primer facie obvious over Yang and Fuser.
Claim 39 recites the method of claim 1, wherein the rootstock comprises nucleic acid encoding two, three, four, five, or more than five guide RNAs.
For the same reason set forth above with respect to claim 1, Yang teaches rootstocks expressing two guide RNAs targeting NIA1 and also two guide RNAs targeting the Venus transgene construct (p958, right column, pa2; p959, left column, pa1). Thus, Yang teaches a rootstock comprising nucleic acid encoding two guide RNAs.
Accordingly, claim 39 is primer facie obvious over Yang and Fuser.
Claim 43 recites the method of claim 1, further comprising retrieving a progeny of the scion, wherein the progeny has an altered genome. Claim 46 recites an edited plant produced by the method of claim 1.
For the same reason set forth above with respect to claim 1, Yang teaches allowing grafted wild-type scions to set seed and screening the progeny for inherited genome edits (p961, left column, pa3-right column, pa1). Thus, Yang teaches retrieving progeny of the scion, wherein the progeny has an altered genome.
Accordingly, claims 43, and 46 are primer facie obvious over Yang and Fuser.
Claim 47 recites a rootstock comprising: nucleic acid encoding a Cas9 nicks or Cas12 nuclease, and nucleic acid encoding a guide RNA for the Cas9 nicks or Cas12 nuclease, wherein the nucleic acid encoding the guide RNA and the nucleic acid encoding the Cas9 nicks or Cas 12 nuclease are fused to nucleic acid encoding a meristem transport segment (MTS).
Yang teaches a transgenic donor rootstock comprising nucleic acid encoding Cas9 fused to a tRNA-like sequence TLS1 or TLS2 mobility motif and nucleic acid encoding guide RNA fused to a TLS mobility motif (Abstract; Supplementary Data 1). Yang teaches that the TLS1/TLS2 mobility motifs confer long-distance RNA mobility into the shoot apical meristem and enable transport of the Cas9 and guide RNA transcripts from the donor rootstock into the scion meristem, thereby permitting genome editing in meristematic cells (p961, left column, pa2; p963, right column pa1). Thus, Yang’s TLS1/TLS2 mobility motifs constitute meristem transport segments (MTS) as broadly claimed because they are nucleotide sequences fused to the editing reagent transcripts to promoter transport into meristem.
As discussed above, it would have been obvious to substitute the Cas9 nuclease of Yang with the Cas9 nicks of Fuser to obtain a rootstock comprising nucleic acid encoding a guide RNA, wherein the guide RNA and Cas9 nicks transcripts are fused to an MTS.
Accordingly, claim 47 is primer facie obvious over Yang and Fuser.
Claim 2 is rejected under 35 U.S.C. §103 as being unpatentable over Yang (2023), in view of Fuser (2014) as apply to claim 1, and further in view of Anzalone (Andrew V. Anzalone et. a., Nature (2019) Vol 576, pp149-156).
Claim 1 as the teachings of Yang and Fuser are discussed above.
Claim 2 is interpreted as dependent of claim 1.
Claim 2 recites the method of claim 1 wherein the Cas9 nicks or Cas 12 nuclease is associated with a reverse transcriptase or fused to a reverse transcriptase, and wherein the guide RNA comprises at its 3' end a priming site and an edit to be incorporated into the genomic target.
For the same reason set forth above with respect to claim 1, Yang teaches graft-mediated genome editing of a scion using a rootstock expressing a mobile Cas9 editing reagent.
Anzalone teaches a prim editing system comprising a Case nicks (H840A) fused to an engineered reverse transcriptase and a prime editing guide RNA (pegRNA) comprising, at its 3’extension, a primer binding site (PBS) and a reverse transcription template encoding the desired edit (Abstract).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to substitute the Cas9 nuclease of Yang with the known Cas9 nicks-reverse transcriptase prime editor and corresponding pegRNA of Anzalone in Yang’s graft-mobile editing system thereby improving editing precision and expanding the types of edits that could be introduced. A person of ordinary skill in the art would have had a reasonable expectation that the prime editing components would function in Yang’s graft-mobile editing platform.
Accordingly, claim 2 is primer facie obvious over Yang, Fuser and Anzalone.
Claims 11, 34 and 36 are rejected under 35 U.S.C. §103 as being unpatentable over Yang (2023), in view of Fuser (2014) as apply to claim 1, and further in view of He (Yubing He et. al., Journal of Genet Genomics (2017) 20; 44(9): 469–472).
Claim 1 as the teachings of Yang and Fuser are discussed above.
Claims 11, 34 and 36 are interpreted as dependent of claim 1.
Claim 11 recites the method of claim 1, wherein the nucleic acid encoding the Cas9 nicks or Cas12 nuclease and the nucleic acid encoding the guide RNA are provided to the rootstock in the same vector.
For the same reason set forth above with respect to claim 1, Yang teaches expression constructs comprising Cas9-TLS and gRNA-TLS mobile CRISPR components for use in transgenic rootstocks (Abstract).
He teaches a single plant transformation construct comprising a Cas9 expression cassette and a guide RNA expression cassette (p6, Fig 1).
It would have been obvious to provide Yang’s Cas9 and guide RNA sequences on the same construct as taught by He in order to facilitate coordinated delivery and expression of the CRISPR components in the transformed plant cells.
Accordingly, claim 11 is primer facie obvious over Yang and Fuser.
Claim 34 recites the method of claim 1, wherein the nucleic acid encoding the guide RNA and the MTS is located between two ribozyme sequences. Claim 36 recites the method of claim 1, wherein the nucleic acid encoding the guide RNA and the MTS further comprises a hammerhead ribozyme sequence 5' to the nucleic acid encoding the guide RNA and the MTS, and an HDV ribozyme 3' to the nucleic acid encoding the guide RNA and the MTS.
For the same reason set forth above with respect to claim 1, He teaches a ribozyme-guide RNA-ribozyme system, wherein a guide RNA is placed between self-cleaving ribozymes to generate a functional guide RNA transcript (p2, pa4). He further teaches a ribozyme-guide RNA-ribozyme system having a hammerhead ribozyme at the 5’end of the guide RNA and an HDV ribozyme at the 3’end of the guide RNA (p6, Fig. 1).
It would have been obvious to modify Yang’s guide RNA-MTS construct to place the guide RNA-MTS sequence between ribozyme sequences as taught by He, and use hammerhead/HDV ribozyme arrange with Yang’s guide RNA-MTS construct, because He teaches that ribozyme process guide RNA transcripts and allow guide RNA expression from various promoters while producing functional guide RNAs.
Accordingly, claims 34 and 36 are primer facie obvious over Yang, Fuser and He.
Claims 41 and 44 are rejected under 35 U.S.C. §103 as being unpatentable over Yang (2023), in view of Fuser (2014) as apply to claim 1, and further in view of Zetsche (Bernd Zetsche et. al., Cell (2015) 163, pp759–771).
Claim 1 as the teachings of Yang and Fuser are discussed above.
Claims 41 and 44 are interpreted as dependent of claim 1.
Claim 41 recites the method of claim 1, wherein the Cas12 nuclease is selected from the group consisting of Cas12a (Cpf1), Casl2e (CasX), Cas12d (CasY), Cas12h, Cas12i, and Cas12j.
For the same reason set forth above with respect to claim 1, Yang teaches graft-mediated genome editing of a scion using a rootstock expressing mobile Cas/gRNA editing reagents fused to TLS/MTS motifs.
Zetsche teaches Cpf1 (Cas12a) as a single RNA-guided class 2 CRISPR nuclease useful for targeted genome editing (Abstract).
It would have been obvious to substitute Yang’s Cas9 nuclease with the Cas12a/Cpf1 nuclease of Zetsche because Cas12a/Cpf1 was a known RNA-guided CRISPR nuclease alternative to Cas9 for targeted genome editing. A person of ordinary skill would have had reason to use Cas12a/Cpf1 in Yang’s graft-mobile CRISPR system to provide another known CRISPR nuclease platform for targeted editing.
Accordingly, claim 41 is primer facie obvious over Yang, Fuser and Zetsche.
Claim 44 recites the method of claim 1, wherein two or more guide RNAs are encoded by a single precursor RNA, wherein the two or more guide RNAs are each flanked by a direct repeat.
For the same reason set forth above with respect to claim 1, Yang teaches using two guide RNAs to generate deletion edits (p958, right column).
Zetsche teaches that Cas12a/Cpf1 processes its own CRISPR RNA array, wherein multiple guide sequence are encoded in a precursor CRISPR array and are separated/flanked by direct repeats to generate crRNAs (p761, right column, pa3).
It would have been obvious to use the Cas12a/Cpf1 CRISPR array format of Zetsche I Yang’s graft-mobile editing system because Yang teaches using multiple guide RNAs for editing, and Zetsche teaches a known and efficient way to express multiple guide RNAs from a single precursor RNA using direct repeats. A person of ordinary skill would have been motivated to use such a precursor array to simplify expression of multiple guide RNAs in the rootstock.
Accordingly, claim 44 is primer facie obvious over Yang, Fuser and Zetsche.
Claim 5 is rejected under 35 U.S.C. §103 as being unpatentable over Yang (2023), in view of Fuser (2014) as apply to claim 1, and further in view of Zetsche (2015) and Fu (Becky Xu Hua Fu et. al., Nature Microbiology (2019) 4(5): 888–897).
Claim 1 as the teachings of Yang and Fuser are discussed above.
Claim 5 is interpreted as dependent of claim 1.
Claim 5 recites the method of claim 1, wherein the Cas12 nuclease is a Cas12 nicks.
For the same reason set forth above with respect to claim 1, Yang teaches graft-mediated genome editing using mobile Cas/gRNA transcripts fused to TLS/MTS motifs.
Yang does not expressly teaches using Cas12a/Cpf1, nor does Yang teach that the Cas12 nuclease is a Cas12 nicks.
Zetsche teaches Cpf1, also known as Cas12a. Fu teaches that Cpf1/Cas12a can exhibit target-dependent nicks activity (Abstract).
It would have been obvious to substitute Yang’s Cas9 nuclease with the Cas12a/Cpf1 nuclease taught by Zetsche and to use the Cas12a/Cpf1 nicks activity taught by Fu because Cas12a/Cpf1 was a known RNA-guided CRISPR editing reagent, and Fu teaches that Cas12a can function as a nicks. A person of ordinary skill would have had reason to use a Cas12 nicks in Yang’s graft-mobile editing system as an alternative DRISPR editing reagent to generate targeted single-strand breaks.
Accordingly, claim 5 is primer facie obvious over Yang, Fuser, Zetsche and Fu.
Claims 24 and 26 are rejected under 35 U.S.C. §103 as being unpatentable over Yang (2023), in view of Fuser (2014) as apply to claim 1, and further in view of James (Anthony James et. al., Frontiers in Plant Science (2022) Volume 13, pp1-13).
Claim 1 as the teachings of Yang and Fuser are discussed above.
Claim 24 is interpreted as dependent of claim 1.
Claim 24 recites the method of claim 1, wherein the nucleic acid encoding the Cas9 nicks or Cas12 nuclease is operably linked to a promoter, wherein the promoter is active in roots and/or phloem companion cells. Claim 26 recites the method of claim 24, wherein: the promoter is the promoter of a gene selected from the group consisting of Arabidopsis WRKY6, chickpea WRKY31, carrot MYB113, corn GLU1, strawberry RB7-type TIP-2, and banana TIP2-2, or the promoter of an orthologous gene thereof.
For the same reason set forth above with respect to claim 1, Yang teaches grafting a wild-type scion onto a transgenic donor rootstock expressing mobile CRISPR genome-editing reagents, including Cas9-encoding RNA and guide RNA fused to tRNA-like mobility sequences, whereby the genome-editing reagents move from the rootstock into the grafted scion and produce heritable genome editing in the scion. Yang further teaches that the Cas9 and guide RNA constructs are expressed in the transgenic donor rootstock (Abstract, Fig 1-5).
James teaches plant promoters having root-directed activity, including promoters associated with banana TIP2-2 genes (Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date to operably link the nucleic acid encoding the Das nuclease of Yang to a root-active promoter, such as the banana TIP2-2 promoter.
Accordingly, claims 24 and 26 are primer facie obvious over Yang, Fuser and James.
Claim 27 is rejected under 35 U.S.C. §103 as being unpatentable over Yang (2023), in view of Fuser (2014), and James (2022) as apply to claim 1, and further in view of Noll (Gundula A Noll et. al., Plant Molecular Biology (2007) 65(3):285-294).
Claim 24 as the teachings of Yang, Fuser and James are discussed above.
Claim 27 is interpreted as dependent of claim 24.
Claim 27 recites the method of claim 24, wherein the promoter is selected from the group consisting of a promoter from a Flowering Locus T (FT) gene, a promoter from a Fabacean FOR1 gene, a rice tungro bacilliform virus promoter, an RmlC-like cupins superfamily protein promoter, a Commelina yellow mottle virus promoter, a wheat dwarf virus promoter, a sucrose synthase promoter, a glutamine synthetase promoter, a phloem-specific isoform of plasmamembrane H+-ATPase promoter, a JMJ18 promoter, and a phloem protein 2 (PP2) promoter.
For the same reason set forth above with respect to claim 1 and 24, Yang teaches grafting a wild-type scion onto a transgenic donor rootstock expressing mobile CRISPR genome-editing reagents. James teaches plant promoters having root-directed activity, including promoters.
Noll teaches the spatial and temporal regulation of the Fabaceae forisome gene FOR1 in phloem tissue and teaches use of the FOR1 regulatory region/promoter to direct gene expression in the phloem (Abstract).
Accordingly, claim 27 is primer facie obvious over Yang, Fuser, James and Noll.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Amjad A Abraham can be reached at (571)272-7058. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/YANXIN SHEN/Examiner, Art Unit 1663
/WEIHUA FAN/Primary Examiner, Art Unit 1663
Prosecution Insights