DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 November 11, 2025 has been entered.
Claims 1-6, 10-14, 17-19 and 23-24 are cancelled.
Claim 8 is currently amended.
Claims 34-36 are new.
Claims 7-9, 15-16, 20-22 and 25-36 are pending.
Claims 7, 9, 15-16, 20-22 and 25-32 are withdrawn.
Claims 8 and 33-36 are examined.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
All previous objections and rejections not set forth below have been withdrawn.
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 .
Withdrawn Claim Rejection
The rejection of claims 8 and 33 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as failing to comply with the written description requirement for the introduction of new matter rejection is withdrawn in light of the amendment of claim 8.
Claim Objections
Claim 33 is objected to because of the following informalities: A series of singular dependent claims is permissible in which a dependent claim refers to a preceding claim which, in turn, refers to another preceding claim. See MPEP § 608.01(n). Specifically, claim 33 is objected to because it refers to a subsequent claim (claim 35), rather than a preceding claim.
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 8 and 33-36 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 8 as currently amended is broadly drawn to a method for identification of a transactivating CRISPRRNA (tracrRNA) of any unspecified organism, the method comprising:
a) providing under unspecified conditions a first single guide RNA candidate comprising a chimeric non- naturally occurring CRISPRRNA (crRNA) comprising a variable targeting domain capable of hybridizing to a target sequence in the genome of a cell, linked to a first nucleotide sequence representing the sense expression of a candidate tracrRNA naturally occurring in said organism;
b) providing under unspecified conditions a second single guide RNA candidate comprising a chimeric non-naturally occurring crRNA comprising a variable targeting domain capable of hybridizing to a target sequence in the genome of said cell, linked to a second nucleotide sequence representing the sense expression of a candidate tracrRNA naturally occurring in said organism;
c) providing under unspecified conditions to the first and second single guide RNA candidates a Cas endonuclease protein from any unspecified source, wherein said Cas endonuclease protein can form a complex with either the first single guide RNA candidate or the second single guide RNA candidate, wherein said complex is capable of introducing a double strand break into said target sequence, wherein the Cas endonuclease and the candidate tracrRNA are from the same organism; and,
d) identification by unspecified means of the first or second guide RNA candidate and its tracrRNA component that complexes to the Cas endonuclease of (c) and results in cleavage of the target sequence in the genome of said cell.
New claim 35 is drawn to the method of claim 8, wherein the Cas-endonuclease and the candidate tracrRNA are from the same CRISPR-Cas system from said organism.
Claim 33 as currently amended is drawn to the method of claim 35, wherein the CRISPR-Cas system is a type II CRISPR-Cas system
New claim 34 is drawn to the method of claim 33 wherein the type II CRISPR-Cas system is a Type II-A, a Type II-B, or a Type II-C subtype.
New claim 36 is drawn to the method of claim 8, wherein the Cas-endonuclease and the candidate tracrRNA are encoded by the same CRISPR-Cas genomic locus in said organism.
The specification describes a method for the identification of a transactivating CRISPRRNA (tracrRNA) of the organism Brevibacillus laterosporus using a Brevibacillus laterosporus strain SSP360D4 Cas endonuclease encoded by a Cas9 gene (SEQ ID NO:36) from an uncharacterized Type II CRISPR-Cas system, and two Brevibacillus laterosporus strain SSP360D4 single guide RNA (sgRNA) variants (SEQ ID NOs: 47 and 48), wherein the single guide RNA (sgRNA) of SEQ ID NO: 47 results in the cleavage of a target sequence (Examples 5-7, FIGs. 18-21, and Table 7).
The specification does not describe any other method for the identification of a transactivating CRISPRRNA (tracrRNA) from any other organism using any other type of Cas endonuclease wherein either a first or a second guide RNA candidate and its tracrRNA component complexes to the Cas endonuclease and results in cleavage of the target sequence in the genome of said cell.
With respect to Cas endonucleases and tracrRNAs, it was known at the time of filing that a large and diverse group of genes are present numerous species of bacteria that encode Cas endonucleases and tracrRNAs. With respect to the ability of a Cas endonuclease to form a complex with a naturally occurring tracrRNA component to introduce a double strand break into a target sequence, it was known at the time of filing that even Cas endonucleases and tracrRNA components from closely related CRISPR-Cas systems may not be able to form a complex that introduces a double strand break into a target sequence, including CRISPR-Cas systems from the same organism.
See, for example, Fonfara et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 2014 Feb;42(4):2577-90. Epub 2013 Nov 22, who were able to identify Cas9 orthologs in 653 bacterial strains representing 347 bacterial species (page 2580 column 2 last paragraph through page 2581 column 1 second paragraph). Fonfara et al. also teach that S. pyrogenes Cas9 can cleave target DNA in the presence of tracrRNA:crRNA duplexes (dual RNAs) from S. mutans and S. thermophilus, but not in the presence of dual RNAs from other bacterial species having closely related CRISPR-Cas systems, and that dual RNA from S. pyrogenes incubated with Cas9 orthologs from bacterial species having closely related CRISPR-Cas systems can cleave target DNA in the presence of Cas9 from S. pyrogenes, S. mutans and S. thermophilus, but not in the presence of Cas9 from the other bacterial species (paragraph spanning columns 1 and 2 of page 2586; page 2587 Figure 6). Fonfara et al. additionally teach that their results support recent findings showing a lack of interchangeability between CRISPR1 and CRISPR3 Cas9 endonucleases of S. thermophilus DGCC7710 with respect to their ability to bind dual-RNA (page 2586 column 2 first paragraph).
Given the breadth of the claims which encompass methods that utilize any combinations of Cas endonucleases and naturally occurring tracrRNA components wherein the Cas endonuclease and the candidate tracrRNA are from the same organism under unspecified conditions to identify by unspecified means a guide RNA comprising a candidate tracrRNA component that complexes to the Cas endonuclease and results in cleavage of a target sequence, including methods wherein the Cas-endonuclease and the candidate tracrRNA are from the same CRISPR-Cas system including wherein the CRISPR-Cas system is a type II CRISPR-Cas system, including wherein the type II CRISPR-Cas system is a Type II-A, a Type II-B, or a Type II-C subtype, and including wherein the Cas-endonuclease and the candidate tracrRNA are encoded by the same CRISPR-Cas genomic locus in said organism, given that it was known at the time of filing that a large and diverse group of genes are present numerous species of bacteria that encode Cas endonucleases and tracrRNAs, and that even Cas endonucleases and tracrRNA components from closely related CRISPR-Cas systems may not be able to form a complex that introduces a double strand break into a target sequence, including CRISPR-Cas systems from the same organism, and given the extremely limited description of only a single method for the identification of a single transactivating CRISPRRNA (tracrRNA) of a single organism (Brevibacillus laterosporus) using a single Cas endonuclease encoded by a Cas9 gene (SEQ ID NO:36) from an uncharacterized Type II CRISPR-Cas system and two single guide RNA (sgRNA) variants, wherein the single guide RNA (sgRNA) of SEQ ID NO: 47 results in the cleavage of a target sequence, one skilled in the art would not recognize that the applicant was in possession of the claimed invention as a whole at the time of filing on the basis of the description in the specification.
Response to Arguments
Applicant's arguments filed November 11, 2025 have been fully considered but they are not persuasive.
Applicant maintains that possession of the claimed subject matter has been demonstrated because the specification describes the method steps recited in the claims, for example, in Examples 5-7, FIGs. 18-21, and Table 7, and because the specification demonstrates that one is able to identify whether the candidate tracrRNA is able, as part of the guide RNA, to form a complex with the Cas endonuclease protein, and whether the complex is able to guide the Cas endonuclease protein to the target sequence in the genome of the cell, and whether cleavage occurs, indicating the candidate tracrRNA is functional. Applicant maintains, therefore, that one skilled in the art, upon reading the specification's disclosure would reasonably conclude that the Applicant was in possession of the claimed invention at the time of filing.
Applicant's arguments are not persuasive.
Applicant's arguments are not persuasive because the claimed genus of methods is extremely broad and variable, as set forth above, whereas the description in the specification in Examples 5-7, FIGs. 18-21, and Table 7 is extremely limited in scope, describing only a single method for the identification of a single transactivating CRISPRRNA (tracrRNA) of a single organism (Brevibacillus laterosporus) using a single Cas endonuclease encoded by a Cas9 gene (SEQ ID NO:36) from an uncharacterized Type II CRISPR-Cas system and two single guide RNA (sgRNA) variants, wherein the single guide RNA (sgRNA) of SEQ ID NO: 47 results in the cleavage of a target sequence. Accordingly, one skilled in the art would not recognize that the applicant was in possession of the claimed invention as a whole at the time of filing on the basis of the description in the specification.
Claims 8 and 33-36 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method for identification of a transactivating CRISPRRNA (tracrRNA) of Brevibacillus laterosporus using the Brevibacillus laterosporus Cas endonuclease encoded by the Cas9 gene of SEQ ID NO:36, does not reasonably provide enablement for other methods for identification of a transactivating CRISPRRNA (tracrRNA) of other organisms using other Cas endonucleases. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
Claim 8 as currently amended is broadly drawn to a method for identification of a transactivating CRISPRRNA (tracrRNA) of any unspecified organism, the method comprising:
a) providing under unspecified conditions a first single guide RNA candidate comprising a chimeric non- naturally occurring CRISPRRNA (crRNA) comprising a variable targeting domain capable of hybridizing to a target sequence in the genome of a cell, linked to a first nucleotide sequence representing the sense expression of a candidate tracrRNA naturally occurring in said organism;
b) providing under unspecified conditions a second single guide RNA candidate comprising a chimeric non-naturally occurring crRNA comprising a variable targeting domain capable of hybridizing to a target sequence in the genome of said cell, linked to a second nucleotide sequence representing the sense expression of a candidate tracrRNA naturally occurring in said organism;
c) providing under unspecified conditions to the first and second single guide RNA candidates a Cas endonuclease protein from any unspecified source, wherein said Cas endonuclease protein can form a complex with either the first single guide RNA candidate or the second single guide RNA candidate, wherein said complex is capable of introducing a double strand break into said target sequence, wherein the Cas endonuclease and the candidate tracrRNA are from the same organism; and,
d) identification by unspecified means of the first or second guide RNA candidate and its tracrRNA component that complexes to the Cas endonuclease of (c) and results in cleavage of the target sequence in the genome of said cell.
New claim 35 is drawn to the method of claim 8, wherein the Cas-endonuclease and the candidate tracrRNA are from the same CRISPR-Cas system from said organism.
Claim 33 as currently amended is drawn to the method of claim 35, wherein the CRISPR-Cas system is a type II CRISPR-Cas system
New claim 34 is drawn to the method of claim 33 wherein the type II CRISPR-Cas system is a Type II-A, a Type II-B, or a Type II-C subtype.
New claim 36 is drawn to the method of claim 8, wherein the Cas-endonuclease and the candidate tracrRNA are encoded by the same CRISPR-Cas genomic locus in said organism.
The specification discloses a method for the identification of a transactivating CRISPRRNA (tracrRNA) of the organism Brevibacillus laterosporus using a Brevibacillus laterosporus strain SSP360D4 Cas endonuclease encoded by a Cas9 gene (SEQ ID NO:36) from an uncharacterized Type II CRISPR-Cas system, and two Brevibacillus laterosporus strain SSP360D4 single guide RNA (sgRNA) variants (SEQ ID NOs: 47 and 48), wherein the single guide RNA (sgRNA) of SEQ ID NO: 47 results in the cleavage of a target sequence (Examples 5-7, FIGs. 18-21, and Table 7).
The specification does not disclose how to identify a transactivating CRISPRRNA (tracrRNA) from any other organism using any other type of Cas endonuclease wherein either a first or a second guide RNA candidate and its tracrRNA component complexes to the Cas endonuclease and results in cleavage of the target sequence in the genome of said cell.
The full scope of the claimed invention is not enabled because the ability of a Cas endonuclease and a single guide RNA comprising a candidate tracrRNA to form a complex capable of introducing a double strand break into a target sequence is unpredictable, given that even Cas endonucleases and tracrRNA components from closely related CRISPR-Cas systems may not be able to form a complex that introduces a double strand break into a target sequence, including CRISPR-Cas systems from the same organism.
With respect to Cas endonucleases and tracrRNAs, it was known at the time of filing that a large and diverse group of genes are present numerous species of bacteria that encode Cas endonucleases and tracrRNAs. With respect to the ability of a Cas endonuclease to form a complex with a naturally occurring tracrRNA component to introduce a double strand break into a target sequence, it was known at the time of filing that even Cas endonucleases and tracrRNA components from closely related CRISPR-Cas systems may not be able to form a complex that introduces a double strand break into a target sequence, including CRISPR-Cas systems from the same organism.
See, for example, Fonfara et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 2014 Feb;42(4):2577-90. Epub 2013 Nov 22, who were able to identify Cas9 orthologs in 653 bacterial strains representing 347 bacterial species (page 2580 column 2 last paragraph through page 2581 column 1 second paragraph). Fonfara et al. also teach that S. pyrogenes Cas9 can cleave target DNA in the presence of tracrRNA:crRNA duplexes (dual RNAs) from S. mutans and S. thermophilus, but not in the presence of dual RNAs from other bacterial species having closely related CRISPR-Cas systems, and that dual RNA from S. pyrogenes incubated with Cas9 orthologs from bacterial species having closely related CRISPR-Cas systems can cleave target DNA in the presence of Cas9 from S. pyrogenes, S. mutans and S. thermophilus, but not in the presence of Cas9 from the other bacterial species (paragraph spanning columns 1 and 2 of page 2586; page 2587 Figure 6). Fonfara et al. additionally teach that their results support recent findings showing a lack of interchangeability between CRISPR1 and CRISPR3 Cas9 endonucleases of S. thermophilus DGCC7710 with respect to their ability to bind dual-RNA (page 2586 column 2 first paragraph).
In the instant case the specification does not provide sufficient guidance with respect to which specific combinations of Cas endonucleases and naturally occurring tracrRNA components to obtain from which species of organism such that the Cas endonucleases and naturally occurring tracrRNA components obtained from the same organism could be used to form a complex that introduces a double strand break into a target sequence in the genome of a cell, and under which conditions, wherein either a first or a second guide RNA candidate and its tracrRNA component complexes to the Cas endonuclease and results in cleavage of the target sequence in the genome of said cell. Absent such guidance one skilled in the art would have to test guide RNAs comprising a variety of different candidate tracrRNA components and a variety of different Cas endonucleases from a variety of different species of organisms under a variety of different conditions in order to determine which guide RNA and Cas endonuclease combinations obtained from the same organism would function as claimed. Such a trial and error approach to practicing the claimed invention would constitute undue experimentation.
Applicant's arguments filed November 11, 2025 have been fully considered but they are not persuasive.
Applicant respectfully submits that a person of ordinary skill in the art having Applicant's specification would be able to make and use a Cas endonuclease protein and a candidate tracrRNA from the same organism to identify a functional tracrRNA, because the skilled artisan is provided with more than sufficient direction and guidance to be able to identify whether the candidate tracrRNA is able, as part of the guide RNA, to form a complex with the Cas endonuclease protein and whether the complex is able to guide the Cas endonuclease protein to the target sequence in the genome of the cell and whether cleavage occurs, indicating the candidate tracrRNA is functional. Applicant maintains, therefore, that one skilled in the art would be able to identify tracrRNAs without undue experimentation.
Applicant's arguments are not persuasive.
Applicant's arguments are not persuasive because the claimed genus of methods is extremely broad and variable, as set forth above, whereas the amount of direction and guidance provided by the specification in Examples 5-7, FIGs. 18-21, and Table 7 is extremely limited in scope, demonstrating only how to identify a single transactivating CRISPRRNA (tracrRNA) of a single organism (Brevibacillus laterosporus) using a single Cas endonuclease encoded by a Cas9 gene (SEQ ID NO:36) from an uncharacterized Type II CRISPR-Cas system and two single guide RNA (sgRNA) variants, wherein the single guide RNA (sgRNA) of SEQ ID NO: 47 results in the cleavage of a target sequence. The specification does not provide additional direction and guidance with respect to how to identify a transactivating CRISPRRNA (tracrRNA) from other organisms using other type of Cas endonucleases. Such guidance is necessary because the ability of a Cas endonuclease and a single guide RNA comprising a candidate tracrRNA to form a complex capable of introducing a double strand break into a target sequence is unpredictable, given that even Cas endonucleases and tracrRNA components from closely related CRISPR-Cas systems may not be able to form a complex that introduces a double strand break into a target sequence, including CRISPR-Cas systems from the same organism. Absent such guidance it would require undue experimentation to practice the invention as broadly claimed. Accordingly, the full scope of the claimed invention is not enabled.
Remarks
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA E COLLINS whose telephone number is (571)272-0794. The examiner can normally be reached M-F 8:30 am - 5:00 pm.
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/CYNTHIA E COLLINS/Primary Examiner, Art Unit 1662
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