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 .
DETAILED ACTION
Claims 17-28 are pending and examined on the merits.
Specification
2. The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code. See MPEP § 608.01. See, for example, page 14.
The disclosure is objected under 37 CFR 1.821(d) as failing to refer to the sequence by use of the sequence identifier, preceded by “SEQ ID NO:” (page 15. Lines 37-40).
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.
3. Claims 17, 20-26 and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (US Patent Application Publication 2016/0208243) as in view of Malnoy et al. (2016, Frontiers in Plant Science 7:1-9).
Instant claims are drawn to a method for producing plant calli of the genus solanum, wherein the produced plant calli have a targeted alteration in a target sequence that is comprised within two or more gene copies, the method comprising a) providing a plant protoplasts comprising said duplex;b) exposing said target sequence in said protoplast to a Cpf1 protein and a CrRNA comprising a guide sequence for targeting said Cpf1 protein to the target sequence comprised within the two or more gene copies, by introducing into into the plant protoplasts said Cpf1 and crRNA using PEG and divalent cation mediated transformation for transient expression, resulting in the targeted alternation of the at least two gene copies and c) regenerating calli from said protoplast; d) genotyping the regenerated calli to detect the targeted alteration of the two and more gene copies; and e) selecting the calli comprising the targeted alteration of the two and more gene copies and wherein the Cpf1 protein comprises an amino acid sequence having at least 99% identity to SEQ ID NO:7; or wherein said duplext DNA is exposed to said Cpt1 and/or crRNA by introducing into the plant protoplast a nucleic acid construct for the transient expression of said Cpf1 and/or crRNA; or wherein the nucleotide acid construct comprising a nucleotide sequence encoding a crRNA being operably linked to a polII promoter; or wherein the nucleotide acid construct comprising a nucleotide sequence encoding a Cpf1 being operably linked to a 35S promoter; or wherein two or more CrRNA are used; or at least one targeted alteration is introduced in the duplex DNA; or the alteration comprises a deletion and an insertion; or wherein the alteration is biallelic; or wherein the method further comprises a step of regenerating a plant cell/plant comprising the targeted alteration; or wherein the divalent is 0.1-0.6 M Calcium Nitrate; or wherein the PEG is PEG 4000; or wherein nucleotide sequence encoding Cpf1 is codon-optimized for expression in tomato; or wherein Cpf1 polypeptide is transfected into the protoplast.
Zhang et al. teach a method of modifying a target locus of interest, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising a Cpf1 effector protein and one or more nucleic acid components, wherein the Cpf1 effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to a target locus of interest that is 3' of a Protospacer Adjacent Motif (PAM), the effector protein induces a modification of the target locus of interest, wherein the complex comprises(claim 15); or wherein the cell is a plant cell (claim 19); or wherein the target locus of interest is modified by the integration of a DNA insert into the staggered DNA double stranded break (claim 28); or wherein the codon is optimized for gene expression (claim 12); or wherein a Cpf1 CRISPR expression system comprises at least:(a) a nucleotide sequence encoding a guide RNA (gRNA) that hybridizes with a target sequence in a plant, and wherein the guide RNA comprises a guide sequence and a direct repeat sequence, and (b) a nucleotide sequence encoding a Cpf1 protein, wherein components (a) or (b) are located on the same or on different constructs, and whereby the different nucleotide sequences can be under control of the same or different regulatory element operable in a plant cell (paragraph [1078]; or wherein he Cpf1 gene and/or guide RNA are encoded by one or more circular or non-circular DNA molecule(s) which are coupled to one or more CPPs for plant protoplast delivery; wherein transient expression of and thus not integrated into the genome (paragraph [1109]; or wherein PEG is used to promote delivery of the nucleic acid (paragraph [1345]); or wherein DNA encoding Cpf1 is operably linked to 35S promoter(paragraph [1130]); or wherein two or more guide sequence operably linked to polymerase III promoter (paragraph [0856]); or wherein the alteration is biallelic (paragaraph [1272]); or wherein the modification may be insertion or deletion (paragaraph [0211]). Zhang et al. also teach regeneration of plant is obtain from cultured protoplast and plant callus. Zhang et al. also teach using Cpf1 to modify polyploid plant that carry duplicate copies of their genome and thereof affect all copies of the gene (paragraph [1131]). Zhang et al. teach that once all copies of the targeted gene is completed (e.g. a normal point mutation in a diploid cell requires two edits), CRISPR enzyme /Cpf1 can be self inactivated (paragraph [0608]).Zhang et al. teach that the plant Is a tomato plant (paragraph [1200]).
Zhang et al. do not teach optimized PEG and divalent mediated transfection for plant protoplast with Cpf1 and guide RNA; Zhang et al. do not teach using Calcium as bivalent cation or PEG4000; Zhang et al. do not teach c) further regenerating a plant from calli d) genotyping the regenerated calli to detect the targeted alteration of the two and more gene copies; and e) selecting the calli comprising the targeted alteration of the two and more gene copies. Zhang et al. do not teach Cpf1 polypeptide is transfected into the protoplast. Zhang et al. do not teach nucleotide sequence encoding Cpf1 is codon-optimized for expression in tomato. Zhang et al. do not explicitly teach using single guide RNA to targeting two or more gene copies.
Malnoy et al. teach transfection of grapevine and apple protoplast with CRISPR/Cas9 Ribonucleoprotein for target mutagenesis of genome. Malnoy et al. teach PEG 4000 and 0.125M CaCL is used for transfection (page 3, right column).
Given the value of using Cpf1/gRNA system for gene editing, it would be obvious for skilled in the art to apply the system of the Cpf1/gRNA of Zhang et al. to plant cell using the transfecting method as taught by Malnoy et al., resulting in instant invention. One would have been motivated to do so given the teaching of Zhang et al. that the Cpf1/gRNA system can be used for a plant cell. It would have been obvious for skilled in the art to replace the 0.125 M CaCL with 0.125 M Calcium nitrate given it would generate a solution with same Calcium concentration.
It would have also been obvious for skilled in the art to further apply modified method to tomato plant given the teaching of Zhang et al. that Cfp1 effector is optimized for expression (claim 16) and that Cpt1 system can be used for modification of the genome of tomato plant (paragraph [1065]).
Although Zhang et al. do not explicitly teach using single guide RNA to targeting two or more gene copies, when single guide RNA was used together with Cpf1 to target a single gene in any multiploidy plant as taught by Zhang et al., it would obviously simultaneously modify other duplicated copies on other set(s) of chromosomes given that they share the same target sequence.
Although Zhang et al. do not teach c) further regenerating a plant from calli d) genotyping the regenerated calli to detect the targeted alteration of the two and more gene copies; and e) selecting the calli comprising the targeted alteration of the two and more gene copies, such steps would have been obvious to be performed given the teaching of Zhang et al. that once all copies of the targeted gene is completed (e.g. a normal point mutation in a diploid cell requires two edits), CRISPR enzyme /Cpf1 can be self inactivated (paragraph [0608]). Therefore, genotyping the regenerated calli to detect the targeted alteration of the two and more gene copies; and e) selecting the calli comprising the targeted alteration of the two and more gene copies would have been performed to ensure that all copies of the targeted gene is completed.
Although the combined teachings do not teach Cpf1 having at least 99% sequence identity to instant SEQ ID NO:7, such Cpf1 is considered as an obvious variants in view of SEQ ID NO:236 of Zhang et al., which share 98.4% identity to instant SEQ ID NO:7, given that making conserved substitutions of residues are well known in the art without changing the function of the protein.
Sequence alignment SEQ ID NO:7 vs. US-14-975-085-236
result Query Filing
No. Score Match Length ID Date Dups Description
-------------------------------------------------------------------------------------------------------------
45 6813 98.4 1352 US-14-975-085-236 2015-12-18 38 NOVEL CRISPR ENZYMES AND SYSTEMS
ALIGNMENT:
Query Match 98.4%; Score 6813; Length 1352;
Best Local Similarity 99.1%;
Matches 1311; Conservative 2; Mismatches 10; Indels 0; Gaps 0;
Qy 1 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKT 60
Qy 61 YADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 YADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDA 120
Qy 121 INKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVF 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 INKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVF 180
Qy 181 SAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEV 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEV 240
Qy 241 FSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPH 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 FSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPH 300
Qy 301 RFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 RFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID 360
Qy 361 LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINL 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINL 420
Qy 421 QEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHL 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 QEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHL 480
Qy 481 LDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTL 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 LDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTL 540
Qy 541 ASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPD 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 ASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPD 600
Qy 601 AAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYA 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 AAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYA 660
Qy 661 KKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYH 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 KKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYH 720
Qy 721 ISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 ISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK 780
Qy 781 LNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSD 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 LNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSD 840
Qy 841 EARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHP 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 EARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHP 900
Qy 901 ETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSV 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 ETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSV 960
Qy 961 VGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLI 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 VGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLI 1020
Qy 1021 DKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFV 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 DKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFV 1080
Qy 1081 DPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVF 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 DPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVF 1140
Qy 1141 EKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNIL 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 EKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNIL 1200
Qy 1201 PKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPM 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 PKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPM 1260
Qy 1261 DADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRGSHHHHHHKLP 1320
||||||||||||||||||||||||||||||||||||||||||||||| | : :
Db 1261 DADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNKRPAATKKAGQAK 1320
Qy 1321 KKK 1323
|||
Db 1321 KKK 1323
4. Claims 1-26 and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (US Patent Application Publication 2016/0208243) as in view of Malnoy et al. (2016, Frontiers in Plant Science 7:1-9) as applied to claims 17, 20-26 and 28 above, and further in view of Doudna et al. (US Patent Application Publication No. 2018/0044700).
Claims 17, 20-26 and 28 are discussed above.
Claims 18-19 further comprise the limitations that the method further comprises a step of synchronizing the cell cycle phase of the protoplast before step (b) or that cell cycle phase is synchronized in S-phase.
Zhang et al. in view of Malnoy et al. do not explicitly teach that the method further comprises a step of synchronizing the cell cycle phase of the protoplast before step (b) or that cell cycle phase is synchronized in S-phase.
Doudna et al. teach a method of site-specific modification of a target DNA comprising blocking the cell at a desired phase in the cell cycle and contacting the target DNA with Cas 9 protein and a guide RNA (claim 1); or wherein the desired phase is S-phase of the cell cycle (claim 2).
It would have been obvious to add an additional step taught by Doudna et al. to synchronize the cell in S-phase before exposure to gene editing system, resulting in instant invention. One would have been motivated to do so given that such method would enhance efficiency of homology-directed repair (HDR) (paragraph [0345]) and teaching of Zhang et al. that Cpf1 may be repaired via HDR ([0014].
5. Claims 17 and 20-28 rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (US Patent Application Publication 2016/0208243) as in view of Malnoy et al. (2016, Frontiers in Plant Science 7:1-9) as applied to claims 17, 20-26 and 28 above, and further in view of Shukla et al. (US Patent Application Publication No. 2013/0326725).
Claims 17, 20-26 and 28 are discussed above.
Claims 27 further comprises the limitations that the method further comprised a step of adding an alginate solution to the protoplast after transformation.
.Zhang et al. in view of Malnoy et al. do not explicitly teach adding an alginate solution to the protoplast after transformation.
Shukla et al. teach that for regeneration of tomato plants, the transformed protoplasts was resuspended in alginate solution to promote callus development (paragraph [0196]).
It would have been obvious for skilled in the art to add such step given the teaching of Shukla that such step would promote callus development.
Conclusion
Claims 17-28 are rejected.
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/LI ZHENG/Primary Examiner, Art Unit 1662
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