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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 Dec. 31, 2025 has been entered.
Claim Status
Claims 25-31 and 33-39 are currently pending in this application.
Election/Restrictions
Applicant’s election of the Cas12 protein species of SEQ ID NO:4 (Cas12p) is acknowledged from the submission filed Dec. 30, 2024. Thus, claims 28 and 30-31 are withdrawn pursuant to MPEP 821. Claims 25-27, 29, and 33-39 have been considered on the merits and all arguments have been fully considered.
Claim Objections
Claims 25-27 are objected to because of the following informalities:
In each of claims 25-27, periods occur after steps ‘a’ and ‘b’, e.g. “a.”. While there is no set statutory form for claims, the present Office practice is to insist that each claim begins with a capital letter and ends with a period. Periods may not be used elsewhere in the claims except for abbreviations. See Fressola v. Manbeck, 36 USPQ2d 1211 (D.D.C. 1995). Applicant should replace “a.” with “(a)”, or “a)”, for example.
Regarding claims 26-27, where a claim sets forth a plurality of elements or steps, each element or step of the claim should be separated by a line indentation, 37 CFR 1.75(i). See MPEP §608.01(m). In each of these claims there appears to be an element ‘a.’ and ‘b.’. Appropriate correction is required.
Claim 33 or 34 recites the phrase “sequence of a human” or “sequence of a non-human primate,” respectively, which would be clearer if rewritten as “a human sequence” or “human genomic sequence” and, respectively, “a non-human primate sequence” or “non-human primate genomic sequence.”
Claim Interpretation
In the claims, the terms “Cas12a.1”, “Cas12p”, and “Cas12q” with regard to modifying the term “gRNA” are interpreted to require the gRNA be capable of forming a functional complex respectively with the respective Cas12a.1”, “Cas12p”, or “Cas12q” protein as defined by claim 25 so as to function to direct the complex to bind and align on a specific target DNA sequence.
In the claims, the phrase “capable of cleaving the target DNA” is interpreted as meaning capable of cleaving at least one phosphodiester bond of the target’s DNA backbone, i.e., endonuclease activity (see instant abstract, [00237]), which requires nickase activity of at least one strand, at least during a first enzymatic transition state (the nickase transition state) (see ). Note, the term “DNA” or “target DNA” encompasses both single-stranded and double-stranded DNAs as well as the DNA component of a DNA-RNA hybrid (see instant [0060], FIG. 17, 26-27).
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 25-31, 35-37, and 39 are rejected under 35 U.S.C. 101 because the claimed invention is not directed to patent eligible subject matter. Based upon an analysis with respect to the claim as a whole, claims do not recite something significantly different than a judicial exception. The rationale for this determination is explained below.
The claims are directed to:
An engineered system comprising:
(a) Cas12 protein as specifically recited, or a nucleic acid encoding it; and
(b) Cas12 gRNA;
wherein the gRNA and the Cas12 protein, or the nucleic acids encoding the aforementioned, do not naturally occur together.
In the preamble, the term “engineered” in view of the instant specification does not imply any limitation to the structures of either the protein or gRNA, nor to a nucleic acid encoding it. Thus, the claims are directed to the combination of two separate nature-based products, (1) the Cas12 protein or nucleic acid encoding it, and (2) its cognate gRNA and nothing more beyond being brought together.
Instant Example 1 of the specification discloses that Cas12a.1, Cas12p and Cas12q encoding nucleic acid sequences were identified from naturally occurring genomic sequences adjacent to cognate gRNA encoding CRISPR arrays (e.g., derived from a Candidatus Micrarchaeota or Candidatus Peregrinibacteria species) ([00129]-[00130]). Regarding claim 30, the naturally occurring SEQ ID NO: 4 comprises SEQ ID NO: 222. The gRNA component as drafted in the instant claims encompasses completely naturally occurring gRNAs, e.g., in a different bacterial strain or isolate.
This nature-based product comprising two different products of nature is analyzed to determine whether it has markedly different characteristics from any naturally occurring counterpart(s) in their natural state. In this regard, the claims require the gRNA must be capable of hybridizing to a target DNA and forming a complex with said cognate Cas12 protein while the Cas12 protein must be capable of cleaving said target DNA (i.e., a catalytic activity).
All of these functional limitations are present in naturally occurring Cas12 proteins and gRNAs. Regarding claim 35, the prior art teaches naturally occurring gRNAs from bacteria (e.g., a Candidatus Peregrinibacteria species) typically target sequences found in bacteria. Regarding claims 37 and 39, the prior art teaches all known natural Cas12 proteins cleave downstream of their target sequence (Li et al., Mol Cell 82: 333-47 (2022) at pg. 335, right col.; Fig. 1A), which inherently involves a nickase activity as explained above.
Thus, the claims encompass two separate molecules that are each identical (no difference in characteristics) to naturally occurring counterparts as claimed. The only difference is these two molecules are combined together into one system, which does not occur in nature. However, there is no evidence that merely bringing them together results in any markedly different characteristic, e.g., regarding target DNAs or DNA cleavage, and thus are a “product of nature” exception. In re Roslin Institute (Edinburgh), 750 F.3d 1333, 1338-39 (Fed. Cir. 2014). Accordingly, the claimed invention is directed to an exception. The mere combination of two or more nature-based products that do not naturally occur together does not automatically confer patent subject matter eligibility to the combination. For example, see the USPTO Subject Matter Eligibility Guidance, Example 6. Bacterial Mixtures (claim 1), Example 10. Food (claim 1), Example 7. Nucleic Acids (claim 1), and Example 5. Genetically Modified Bacterium (claim 1).
Because the claimed invention does not include any additional features that could add significantly more to the exception, the claimed culture does not qualify as eligible subject matter, and should be rejected under 35 U.S.C. § 101 as explained below. The gRNA component’s closest natural counterparts functions as it would in nature, such as if it ever encountered the Cas12 protein component closes natural counterpart and vice versa. Although the two components may form a non-natural complex (i.e., have the capability of forming a complex), none of the claims require this structure is formed. Instead dependent claim 27 emphasizes how claim 25 encompasses only nucleic acid components which cannot interact to form any non-naturally occurring structure. Similarly, claim 25 encompasses wherein the Cas12 component is a protein but the gRNA component is a DNA encoding said gRNA, which again cannot interact with the Cas12 protein to form a non-naturally occurring structure.
An examination of Step 2A prong 2, the answer is no because the claimed invention does not integrate the judicial exception, in the instant case a system, into a practical application. It is only the recited limitations in the claims that are examined under 101 and not aspects such as what the system is used for. It is emphasized that the claimed invention is a product and not a process of using or making a product.
An examination of Step 2B, the answer is no with respect to the claimed invention. There are no other additional elements recited in the instant claims that would amount to significantly more than the judicial exceptions. Each of the two molecules as claimed are indistinguishable from those that exist in nature and there are no limitations that add any additional elements to the claimed compositions. Combining these two molecules into a single system does not appear to change either molecule (1) or (2) in any significant or meaningful way to amount to more than the judicial exception.
Claim Rejections - 35 USC § 112(a), Written Description
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 25-27, 29 and 33-39 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.
When claim 25 is analyzed in light of the specification, the instant invention is directed to a system comprising (a) a Cas12 protein selected from Cas12a.1, Cas12p, and Cas12q or, alternatively, a nucleic acid encoding the aforementioned; and (b) a cognate Cas12 gRNA or, alternatively, a nucleic acid encoding the aforementioned. While the sequence identity of the Cas12 protein is limited by the claim, the gRNA is only limited by functional limitations of: (1) not naturally occurring together with the Cas12 protein, (2) capable of hybridizing to a target sequence in a target DNA, and (3) capable of forming a complex with the Cas12 protein. The target sequence is not structurally defined by merely limited functionally as being hybridizing with the gRNA and being comprised within a target DNA.
M.P.E.P. §2163 states “To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116.”
In the instant case, claim 25 broadly comprises a genus of gRNAs defined only functionally as well as a genus of any target sequence in a target DNA bound by the gRNA. The claim is also broad in that (1) the gRNA need not be capable of hybridizing to the target DNA sequence when complexed with the Cas12 protein; and (2) the Cas12 protein is capable of cleaving the target DNA regardless of gRNA complex formation. Further, claim 25 is broad in that the Cas12 protein need only comprise a sequence at least 90% identical to instant SEQ ID NO: 3-5 or 222.
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described.
Claim 25 encompasses a broad genus of Cas12a.1, Cas12p, and Cas12q proteins due to the permitted amino acid sequence variance but functionally restrained to those capable of cleaving a target DNA substrate. However only four representative species are described, one species for each of Cas12a.1 and Cas12q and two variants of Cas12p, consisting exactly of SEQ ID NOs: 3-5 or 222. Based on the prior art, a single amino acid sequence variation in a Cas protein can prevent or inactivate catalytic activity of a nuclease domain, e.g., in the NUC lobe at D10A, E477A, H701A, or D704A, or the PI domain at N985A, N986A, R991A, E993A, or R1015A, with numbering based on S. aureus Cas (US20180312824A1 at FIG. 1, 15, 18; [0361]-[0386]; [0171]).
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, methods of making the claimed product, or any combination thereof. The disclosure of a single species is rarely, if ever, sufficient to describe a broad genus, particularly when the specification fails to describe the features of that genus, even in passing. (see In re Shokal 113USPQ283(CCPA1957); Purdue Pharma L.P. vs Faulding Inc. 56 USPQ2nd 1481 (CAFC 2000).
Here, 90% identity to an over 1,100 residue long protein, encompasses at least 20110 theoretical structural variants, without guidance as to which might exhibit nuclease activity as oppose to those lacking nuclease activity. As there is unpredictability as to which of the at least 90% sequence identity of the provided four species can vary and still retain nuclease activity, the scope of the claims is not adequately described. The same logic applies to dependent claims 36-39.
The instant application lacks a single description of any species of Cas12 protein capable of cleaving a target DNA regardless of gRNA complex formation or of a Cas12 protein that forms a complex with a gRNA whereby the gRNA cannot base pair with the target sequence.
Thus, the skilled artisan cannot envision which, or even how to identify which, Cas12a.1, Cas12p, and Cas12q proteins satisfy the claim limitations of (1) both 90% sequence identity and cleaving a target DNA, (2) forms a complex with a gRNA wherein the complex cannot hybridize to the target DNA. Therefore, there is a lack of evidence in the instant specification as filed that the inventors were in possession of the entire scope of Cas12 proteins encompassed by the claims.
Claims 25-27, 29 and 33-39 encompasses a broad genus of gRNAs without structural limitation. Instead, the gRNA is defined only functionally as capable of (1) hybridizing to a “target” sequence in a “target DNA” and (2) forming a complex with the respective Cas12 protein. As the claims do not specific what constitutes a “target DNA” beyond it must comprise the target sequence, such as one in a DNA of a human, NHP, or associated with any bacteria or virus (claims 33-35), the target DNA could be as small as the target sequence alone (consisting of) or as large as over one million nucleotides (e.g., a human chromosome).
An adequate written description must contain enough information about the actual makeup of the claimed products — "a precise definition, such as by structure, formula, chemical name, physical properties, or other properties, of species falling within the genus sufficient to distinguish the genus from other materials," which may be present in "functional" terminology "when the art has established a correlation between structure and function." Ariad, 598 F.3d at 1350.
In the instant case, the gRNA genus is described and ordinarily understood as a Class 2 Type V guide RNA (gRNA) structurally comprising an unbranched RNA molecule comprising either (1) a crRNA and tracrRNA complex or (2) a single engineered fusion RNA comprising a crRNA/spacer of about 25-50 bases (nt) (e.g., variable targeting spacer sequence or entire crRNA) fused with a “mature scaffold” comprising a specific direct repeat (e.g., SEQ ID NO: 6-8, 19-21 or 26-42) or Cas12 protein binding sequence (e.g., a hairpin duplex or tracrRNA “handle”), wherein the spacer has a chosen/designed targeting sequence complementary to a “target” nucleic acid of at least about 60-99% over 23 contiguous bases at the 5’ terminus, perhaps based on in silico deductions ([00149]-[00159]; FIG. 38). Furthermore, each Cas12 protein is understood to inherently have its own PAM specificity (see [00167]; FIG. 8), which paired with the spacer sequence together determine Cas12 protein-gRNA complex binding to a target nucleic acid (see Mendoza and Trinh, Biotechnol J 13: e1700595 (2018) at pg. 1, right col.).
The term “target sequence” is described broadly as encompassing any polynucleotide having 1-23 nucleotides, such as 8-25 nucleotides, with at least about 1-23 nucleotides having at least 60% sequence identity to a given gRNA spacer ([00150]-[00157]). Therefore, the Cas 12 gRNA structure of the claims encompasses over 250,000 variants (4^23) in the spacer sequence portion alone and the target sequence variants include all possible 279,841 variants (4^23) of any 23 polynucleotide sequence complementary to the spacer sequence as anchored by having just 1-23 nucleotides with at least 60% sequence identity thereto (www.calculator.net/exponent-calculator).
As there is no established correlation between Cas12 gRNA functional abilities and its representative structures (nexus), the instant specification in view of the prior art fails to provide any additional species beyond the instant representative species disclosed. However Applicant is invited to provide evidence to the contrary. How can a given target DNA (structure) and target sequence be determined merely by these functional limitations wherein the target sequence can be any 23mer sequence at least 60% identical to any programmable spacer sequence and the Cas12 protein can vary by as much as 90% sequence identity, conceivably altering PAM specificity in unpredictable ways. Furthermore, the description does not adequately describe the structure of a suitable Cas12 gRNA of the invention having any mature scaffold and crRNA of 25-50 bases, rather a few representative species are described lacking a nexus to the breadth of the functional limitations to the gRNA and target sequence as claimed.
The written description requirement may be satisfied through actual reduction to practice, reduction to drawings, or by disclosure of relevant identifying characteristics, i.e. structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between structure and function, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of any gRNA as functional defined to target virtually any DNA target. In the instant case, the specification fails to provide sufficient descriptive information. The general knowledge and level of skill in the art do not supplement the omitted description because specific, not general, guidance is what is needed. Thus, no identifying characteristics or properties are provided such that one of skill would be able to predictably produce an such gRNA across the full genus encompassed by the claims defined as forming a complex with the specific respective Cas12 protein in the claim.
Vas-Cath Inc. v. Mahurkar, 19USPQ2d 1111, clearly states “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the ‘written description’ inquiry, whatever is now claimed” (pg 1117). The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed” (pg 1116). As discussed above, the skilled artisan cannot envision the gRNA genus even provided with a few examples of how to experimentally find other species, and therefore conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method of isolation. Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method of isolating it. The compound itself is required. See Fiers v. Revel, 25 USPQ2d 1601 at 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016. One cannot describe what one has not conceived. See Fiddes v. Baird, 30 USPQ2d 1481 at 1483. In Fiddes, claims directed to mammalian FGFs were found to be unpatentable due to lack of written description for that broad class. The specification provided only the bovine sequence.
Amgen, Inc., v. Sanofi (2023)
“Amgen seeks to monopolize an entire class of things defined by their function”. “The record reflects that this class of antibodies does not include just the 26 that Amgen has described by their amino acid sequence, but a ‘vast’ number of additional antibodies that it has not.” “It freely admits that it seeks to claim for itself an entire universe of antibodies.”
In the instant case, the record reflects that the claims encompass an enormously vast genus of gRNAs, structurally encompassing over 279,841 spacer sequence variations with at least 3 million (e.g., 434) theoretical structural variants of just the spacer/crRNA component chimerically paired with a given gRNA scaffold, e.g. comprising instant SEQ ID NO: 6-8, 19-21, 26-42, and/or 116-117 ([00154]; [00151]; www.calculator.net/exponent-calculator). These gRNAs function to hybridize to any target DNA comprising a bare minimum of sequence similarity to said 279,841 spacer sequences wherein the target DNA may comprise an additional, unlimited length of sequence, such as including the Cas12 complex cleavage site, or instead may merely consist of the exact complement to the 279,841 spacer sequences and be cleaved internally within. This is the entire universe of gRNAs only functionally limited to form a complex with the three recited Cas12 protein genera.
Accordingly, this limited information is not deemed sufficient to reasonably convey to one skilled in the art that the applicant is in possession of the enormously vast genus of gRNAs structurally unrecited and functionally undisclosed polynucleotides. Thus, for the reasons outlined above, it is concluded that the claims do not meet the requirements for written description under 35 U.S.C. 112, first paragraph. All dependent claims are included in the basis of the rejection because they do not correct the primary deficiencies of the independent claim.
35 USC § 112(a), Scope of Enablement
Claims 25-27, 29, and 33-39 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because while the claims are enabled for wherein the Cas12 protein consists of SEQ ID NO: 3, 4, 5 or 222 and the gRNA comprises SEQ ID NO: 177-181, 225-226, 231-233, or 237-239, and when complexed the gRNA is capable of hybridizing to a target DNA sequence and the complex is capable of cleaving the target DNA; 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 any engineered system comprising any Cas12a1 protein having at least 90% sequence identity to SEQ ID NO: 3 and paired with any Cas12a1 gRNA, any Cas12p protein having at least 90% sequence identity to SEQ ID NO: 4 or 222 and paired with any Cas12p gRNA, or any Cas12q protein having at least 90% sequence identity to SEQ ID NO: 5 and paired with any Cas12q gRNA.
Enablement is considered in view of the Wands factors (MPEP 2164.01 (a)). The court in Wands states that "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue or unreasonable experimentation. The key word is 'undue.' Not 'experimentation;" (Wands, 8 USPQ2d 104). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighting many factual considerations." (Wands, 8 USPQ2d 1404).
The factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation required is “undue” or unreasonable include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
Furthermore, the USPTO does not have laboratory facilities to test if an invention will function as claimed when working examples are not disclosed in the specification. Therefore, enablement issues are raised and discussed based on the state of knowledge pertinent to an art at the time of the invention. And thus, skepticism raised in the enablement rejections are those raised in the art by artisans of expertise.
All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature and breadth of the invention:
The claims are directed to a system comprising (a) a Cas12 protein selected from Cas12a.1, Cas12p, and Cas12q or, alternatively, a nucleic acid encoding the aforementioned; and (b) a Cas12 gRNA or, alternatively, a nucleic acid encoding the aforementioned. The gRNA is only limited by functional limitations of: (1) not naturally occurring together with the Cas12 protein, (2) capable of hybridizing to a target sequence in a target DNA, and (3) capable of forming a complex with the Cas12 protein. The Cas12a.1, Cas12p, and Cas12q proteins are structurally limited by species of reference protein sequences but allowing for 90% amino acid sequence variance and are functionally constrained to those capable of cleaving a target DNA substrate.
In Amgen, the Supreme Court, held that claims drawn to a genus of monoclonal antibodies were invalid due to lack of enablement wherein the genus was merely functionally claimed by their ability to bind to a specific protein as opposed to reciting a specific structure. MPEP 2164.01 explains "it may suffice to give an example (or a few examples) if the specification also discloses some general quality . . . running through the class that gives it a peculiar fitness for the particular purpose" and "disclosing that general quality may reliably enable a person skilled in the art to make and use all of what is claimed, not merely a subset." Id. at 611 (internal quotations omitted). However, while the specification in Amgen identified 26 exemplary antibodies that performed the claimed function by their amino acid sequences, the claims at issue were directed to a class which included "a ‘vast’ number of additional antibodies" that Amgen had not described by their amino acid sequences. Id. at 613. The Court found that the patent owner sought to monopolize an entire class by their function, even though that class was much broader than the exemplary antibodies disclosed by their amino acid structure. Here, the gRNA described with a high level of generality in only functional terms resembles the situation in the Amgen case and as noted above may encompass millions of undescribed and unrecited gRNA structures.
The state of the art:
Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are naturally occurring in the genomes of bacteria and archaeal species as CRISPR arrays comprising repeating units interspersed with spacers and an adjacent Cas gene comprising conserved nuclease catalytic domains (Ishino et al., J Bacteriol 200: e00580-17 (2018) at pg. 8, Fig. 4). The prior art teaches scientists, predominantly via bioinformatics and then empirical validation, successfully identified at least thirty Cas proteins based on the foundational spCas9 by processes which overtime became increasingly more efficient (Ishino et al., (2018) at pg. 5, para. 3-4; pg. 6, para. , to pg. 9; Fig. 6; Murugan et al., Mol Cell 68(1): 15-25 (2017) at pg. 2, para. 2-4; pg. 3, last para., to pg. 7). For example, by 2018 scientists had identified multiple Cas protein families, including a dozen Cas9 proteins (e.g., NmCas9, SaCas9, CjCas9, etc.) along with Cas3, Cas14, and multiple Cas12 and Cas13 family members, such as Cas12a, Cas12b, Cas12c, and CasX, representing at least 2 classes, 6 types, and 22 subtypes (Cas1-Cas10) (see e.g., Li et al., Mol Cell 82: 333-47 (2022) at Suppl. table 1; Mendoza and Trinh (2018) at pg. 1, right col., 2nd para.). In particular, Cas12b (C2c1), Cas12c, Cas12(d) (CasY) and Cas12e (CasX) were discovered by computational prediction approaches and genomic sequence databases based on previously characterized Cas system sequences (Shmakov et al., Mol Cell 60: 385-97 (2015) at Fig. 1B, pg. 386; Burnstein2 (Burnstein et al., Nature 542: 237-41 (2017) at pg. 237, Fig. 1-2; Yan et al., Science 363: 88-91 (2019) at pg. 1, col. 1-2, Fig. 1).
The prior art teaches all gRNAs are small polyribonucleotides having a particular sequence, thusly all gRNAs are capable of hybridizing to certain “target” DNA molecules in certain situations, e.g., one comprising a reverse complementary sequence, which could be categorized as a “target” sequence. The prior art teaches that Cas endonuclease target recognition and nuclease activity both depend on the Cas protein and its cognate gRNA forming a complex as well efficient cleavage depending on the spacer region of the gRNA forming a heteroduplex with the target (Lim et al., Nat Commun 7: 13350 (2016) at abstract; Fig. 5). For example, Cas12 (Cpf1) can cleave DNA in certain situations in the absence of any RNA, but this cleavage is not sequence specific (Sundaresan et al., Cell Rep 21: 3728-39 (2017) at summary). Thus, there is no evidence in the prior art of a Cas12 protein capable of cleaving a target DNA without gRNA complex formation nor wherein the Cas12-gRNA complex cannot bind to its PAM/gRNA target sequence.
While the prior art reveals some structure-function correlations between Cas9 and gRNA functional abilities (Jiang and Doudna, Annu Rev Biophys 46: 505-29 (2017) at Fig. 3-5), the instant specification purports to have newly discovered three Cas12 proteins (Cas12a1, Cas12p and Cas12q) wherein such research couldn’t have existed before the effective filing date and, thus, any expansion/envisaging of gRNA structures beyond what is instantly taught relies on the data and species disclosed in the instant application, which are limited in number as detailed below.
The amount of direction and guidance and working examples provided by Applicant:
The instant application provides no evidence of any Cas12 protein cleaving a DNA substrate in a targeted manner in the absence of an appropriate gRNA, i.e., a cognate gRNA, whereby a Cas12-gRNA complex forms that together binds and cleaves a target DNA in a sequence-specific manner.
The instant application provides a limited number of working examples of gRNA species (e.g., SEQ ID NO: 177-181, 225-226, 231-233, and 237-239), all of which comprise either a full or portion of a natural direct repeat sequence, optionally having two specific point mutations in the stem-loop region to improve stability (Table 2, 5a-b; Example 1, FIGs 5B, 5D, 5F, 7A-C). While one of skill in the art could use this information to design a great diversity of spacer sequences without undue experimentation, to extrapolate these species to the scope of undisclosed species encompassed by the claims would require undue experimentation (regarding non-spacer sequence structure variances); however, Applicant is invited to provide evidence to the contrary.
Extensive experimentation would be required to determine the functional pairings over the scope of Cas12a1, Cas12p, or Cas12q protein having at least 90% sequence identity to SEQ ID NO: 3, 222, or 5, respectively, with any gRNA defined only functionally and without structural limitation. As noted above there are over 20110 theoretical structural variants for each of Cas12a1, Cas12p, or Cas12q that may be tested for functionality.
In summary, claims 25-27, 29, and 33-39 are rejected under 35 U.S.C. 112(a) because the specification does not reasonably provide enablement, to a person skilled in the art to which it pertains or with which it is most nearly connected to, to make and use the system comprising said Cas12 protein (or nucleic acid encoding it) and said gRNA (or nucleic acid encoding it). The limited guidance provided in the specification, the lack of guidance in the prior art regarding Cas12a.1, Cas12p and Cas12q, and the broad scope of the claims with regard to gRNA structure; undue and unreasonable experimentation would have been required for one skilled in the art to achieve targeted DNA cleavage across the entire scope of the claim terms: Cas12a.1, Cas12p, and Cas12q gRNA, which must work concordantly both in structure and function with the “90% identity genus” respectively of the four recited Cas12 proteins for this functional achievement.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(B) CONCLUSION. —The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 37 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
Claim 37 recites the relative term “distal” for the position of a cleavage site relative to a target sequence bound by the Cas12-gRNA complex. This relative term renders the claim indefinite because neither the claim nor the specification provides a definition or standard distance range for “distal” or direction (e.g., 5’ or 3’) and, thus, one of ordinary skill in the art would not be reasonably appraised of the scope of this limitation (see MPEP 2173.05(b)).
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 36-39 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 36 further recites wherein the Cas12 protein of claim 25 is catalytically active; however, claim 25 requires the Cas12 protein be capable of cleaving the target DNA, which implies a catalytic activity. Thus, claim 36 fails to further limit the subject matter of a claim 25.
Claim 37 further limits the cleavage to sites distal to the target sequence. As the prior art teaches all known Cas12 proteins cleave downstream of their target sequence (Li et al., Mol Cell 82: 333-47 (2022) at pg. 335, right col.; Fig. 1A), claim 37 prima facie fails to further limit the subject matter of claim 25 and 36 absence evidence to the contrary.
Claim 38 further recites wherein the Cas12 protein of claim 25 is catalytically dead; however, claim 25 requires the Cas12 protein be capable of cleaving the target DNA, which implies a catalytic activity. Thus, claim 38 fails to include all the limitations of claim 25.
Claim 39 further recites wherein the Cas12 protein of claim 25 comprises nickase activity; however, claim 25 requires the Cas12 protein be capable of cleaving the target DNA (such as an ssDNA), which implies a catalytic nickase activity as present in naturally occurring Cas12 proteins. Thus, claim 39 fails to further limit the subject matter of a claim 25.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 25-27 and 29 are rejected under 35 U.S.C. 103 as being unpatentable over Koonin (Koonin et al., Curr Opin Microbiol 37:67-78 (2017); IDS ref.) in view of Murugan (Murugan et al., Mol Cell 68: 15-25 (2017)) and Couvin (Couvin et al., Nucleic Acids Res 46: W246-51 (2018)), and as evidenced by KU516160 (NCBI GenBank Accession KU516160, submitted 08-MAR-2016) and KKQ38174 (NCBI GenBank Accession KKQ38174, submitted 07-MAY-2015).
The claims are interpreted as provided in a previous section.
Koonin teaches multiple Cas12 CRISPR/Cas systems (e.g., Cas12a-e) were already known and had been used to engineer sequence programmable induction of double-stranded DNA breaks (DSBs) with high efficiencies by forming complexes with synthetic gRNAs (Fig. 1, 3; pg. 71, left col., last para., to pg. 73, right col., 2nd para.). Koonin teaches specific Cas12 variants include Cas12a (Cpf1), Cas12b (C2c1), Cas12c, Cas12d, and Cas12e (Fig. 1, 3), with known protein sequences which cleave dsDNA via RuvC activity and sequence targeting by the gRNA’s spacer when complexed (Table 1; Fig. 1). Koonin teaches methods of searching databases to identify new Cas12 variants using in silico software analysis of prokaryotic genomic sequences anchored by the sequence of a known Cas gene(s) (pg. 71, left col., last para., to right col., 2nd para.).
Regarding claims 25-27 and 29, Koonin does not teach wherein the Cas12 protein comprises an amino acid sequence having at least 90% sequence identity to any one of instant SEQ ID NOs: 3-5 or 222.
However Murugan teaches a desire for new gene-editing Cas protein tools with different characteristics for use in creating new CRISPR/Cas engineering tools differentiated from ones already in use, such as characteristics regarding different PAM specificities or broader PAM tolerances and different nuclease activities, e.g., creation of directional sticky ends (pg. 2, 4th para., pg. 10-12; pg. 10, last two para.).
Couvin teaches the availability of CrisprCasFinder software, having parameter settings for finding Class 2 type V Cas sequences and/or those data sets including known Cas12 proteins (pg. W249). Moreover, websites of the U.S. National Center for Biotechnology Information (NCBI), e.g., UniRef and GenBank, taught genomes and genomic sequences of various prokaryotes encoding various Cas12 variants, such as Candidatus Micrarchaeota and Candidatus Peregrinibacteria as noted by instant Example 1.
It would have been prima facie obvious to one of ordinary skill in the art before the earliest effective time of filing to identify new Cas12 protein variants, or a nucleic acid sequence encoding such variant, having any sequence readily searchable in an NCBI database publicly available before the instant filing date, such as a “Cas12p” comprising SEQ ID NO: 4, and then use laboratory tests to confirm DNA cleavage activity, such as in vitro, using routine assays comprising a naturally occurring gRNA of the same species as the Cas12 protein but retargeted with a spacer sequence matching a test DNA substrate sequence (e.g., a reporter). One of ordinary skill in the art would be motivated to search and screen for alternative Cas12 proteins to expand the diversity of CRISPR/Cas genomic editing tools. One would have a reasonable expectation of success to specifically find and create nucleic acids encoding a Cas12p consisting of SEQ ID NO: 4 based on known sequences of other Cas12 proteins, such as Cas12a (Cpf1, PDB:5B43), Cas12b (C2c1, PDB:5WQE, PDB:5NG6), Cas12c, Cas12d, and/or Cas12e discussed in Koonin and with searchable databases comprising genomic encoding sequences known respectively in Francisella cf. novicida (FnCas12a, FNFX1_1431-FNFX1_1428), Acidaminococcus sp. (AsCas12a), Alicyclobacillus acidoterrestris (Aac-Cas12b, N007_06525-N007_06535), Oleiphilus sp. (A3715_16885-A3715_16890), Bacterium CG09_39_24 (BK003_02070-BK003_02075), and Deltaproteobacteria sp. (A2Z89_08250-A2Z89_08265) (Fig. 1). Further, it would have been prima facie obvious to one of ordinary skill in the art before the earliest effective time of filing to identify new Cas12 protein variants using available software tools for prior art purposes, including CrisprCasFinder taught by Couvin and/or methods taught by Koonin.
For example as evidenced by KU516160 or KKQ38174 before the earliest effective filing, a CRISPR/Cas system nucleic acid sequence of a metagenomic study comprising unidentified bacteria isolated from groundwater (KU516160) and the hypothetical Cas protein of Candidatus Roizmanbacteria bacterium (KKQ38174) were each known to relate to a Cas12 variant protein similar to instant SEQ ID NO: 4, i.e., with over 79% similarity and over 63% sequence identity, as shown below.
SEQ ID 4 6 FDQFVNQYALSKTLRFELKPVGETGRMLEEAKVFAKDETIKKKYEATKPFFNKLHREFVE
KU516160 4 FDSFTNLYSLSKTLKFEMRPVGNTQKMLDNAGVFEKDKLIQKKYGKTKPYFDRLHREFIE
** * * * ***** ** *** * ** * ** ** * *** *** * ***** *
SEQ ID 4 66 EALNEVELAGLPEYFEIFKYWKRYKKK--FEKDLQKKEKELRKSVVGFFNAQAKEWAK-K
KU516160 64 EALTGVELIGLDENFRTLVDWQKDKKNNVAMKAYENSLQRLRTEIGKIFNLKAEDWVKNK
*** *** ** * * * ** * ** ** * * * *
SEQ ID 4 123 YETLGVKKKDVGLLFEENVFAILKERYGNEEGSQIVDES---TGKD----VSIFDSWKGF
KU516160 124 YPILGLKNKNTDILFEEAVFGILKARYGEEKDTFIEVEEIDKTGKSKINQISIFDSWKGF
* ** * * **** ** *** *** * * * *** *********
SEQ ID 4 176 TGYFIKFQETRKNFYKDDGTATALATRIIDQNLKRFCDNLLIFESIRDKIDFSEVEQTMG
KU516160 184 TGYFKKFFETRKNFYKNDGTSTAIA TRIIDQNLKRFIDNLSIVESVRQKVDLAETEKSFS
**** ** ******** *** ** ************ *** * ** * * * * *
SEQ ID 4 236 NSIDKVFSVIFYSSCLLQEGIDFYNCVLGGETLPNGEKRQGINELINLYRQKTSE-KVPF
KU516160 244 ISLSQFFSIDFYNKCLLQDGIDYYNKIIGGETLKNGEKLIGLNELINQYRQNNKDQKIPF
* ** ** **** *** ** ***** **** * ***** *** * **
SEQ ID 4 295 LKLLDKQILSEKEKFMDEIENDEALLDTLKIFRKSAEEKTTLLKNIFGDFVMNQGKYDLA
KU516160 304 FKLLDKQILSEKILFLDEIKNDTELIEALSQFAKTAEEKTKIVKKLFADFVENNSKYDLA
*********** * *** ** * * * * ***** * * *** * *****
SEQ ID 4 355 QIYISRESLNTISRKWTSETDIFEDSLYEVLKKSKIVSASVKKKDGGYAFPEFIALIYVK
KU516160 364 QIYISQEAFNTISNKWTSETETFAKYLFEAMKSGKL--AKYEKKDNSYKFPDFIALSQMK
***** * **** ****** * * * * * * *** * ** **** *
SEQ ID 4 415 SALEQIPTE-KFWKERYYKNIGDVLNKGFLNGKEGVWLQFLLIFDFEFNSLFEREIIDEN
KU516160 422 SALLSISLEGHFWKEKYYK-ISKFQEKTN-------WEQFLAIFLYEFNSLFSDKINTKD
*** * * **** *** * * * *** ** ****** *
SEQ ID 4 474 GDKKVAGYNLFAKGFDDLLNNFKYD--QKAKVVIKDFADEVLHIYQMGKYFAIEKKRSWL
KU516160 474 GETKQVGYYLFAKDLHNLILSEQIDIPKDSKVTIKDFADSVLTIYQMAKYFAVEKKRAWL
* * ** **** * * ** ****** ** **** **** **** **
SEQ ID 4 532 ADYDIDSFYTDPEKGYLKFYENAYEEIIQVYNKLRNYLTKKPYSEDKWKLNFENPTLADG
KU516160 534 AEYELDSFYTQPDTGYLQFYDNAYEDIVQVYNKLRNYLTKKPYSEEKWKLNFENSTLANG
* * ***** * *** ** **** * ***************** ******** *** *
SEQ ID 4 592 WDKNKEADNSTVILKKDGRYYLGLMARGRNKLFDDRNLPKILEGVENGKYEKVVYKYFPD
KU516160 594 WDKNKESDNSAVILQKGGKYYLGLITKGHNKIFDDRFQEKFIVGIEGGKYEKIVYKFFPD
****** *** *** * * ***** * ** **** * * * ***** *** ***
SEQ ID 4 652 QAKMFPKVCFSTKGLEFFQPSEEVITIYKNSEFKKGYTFNVRSMQRLIDFYKDCLVRYEG
KU516160 654 QAKMFPKVCFSAKGLEFFRPSEEILRIYNNAEFKKGETYSIDSMQKLIDFYKDCLTKYEG
*********** ****** **** ** * ***** * *** ********* ***
SEQ ID 4 712 WQCYDFRNLRKTEDYRKNIEEFFSDVAMDGYKISFQDVSESYIKEKNQNGDLYLFEIKNK
KU516160 714 WACYTFRHLKPTEEYQNNIGEFFRDVAEDGYRIDFQGISDQYIHEKNEKGELHLFEIHNK
* ** ** * ** * ** *** *** *** * ** * ** *** * * **** **
SEQ ID 4 772 DWN--EGANGK-----KNLHTIYFESLFSADNIAMNFPVKLNGQAEIFYRPRTEGLEKER
KU516160 774 DWNLDKARDGKSKTTQKNLHTLYFESLFSNDNVVQNFPIKLNGQAEIFYRPKTE---KDK
*** ** ***** ******* ** *** ************ ** *
SEQ ID 4 825 IITKKGNVLEKGDKAFHKRRYTENKVFFHVPITLNRTKKNPFQFNAKINDFLAKNSDINV
KU516160 831 LESKKD---KKGNKVIDHKRYSENKIFFHVPLTLNRTKNDSYRFNAQINNFLANNKDINI
** ** * ** *** ***** ****** *** ** *** * ***
SEQ ID 4 885 IGVDRGEKQLAYFSVISQRGKILDRGSLNVINGVNYAEKLEEKARGREQARKDWQQIEGI
KU516160 888 IGVDRGEKHLVYYSVITQASDILESGSLNELNGVNYAEKLGKKAENREQARRDWQDVQGI
******** * * *** * ** **** ********* ** ***** *** **
SEQ ID 4 945 KDLKKGYISQVVRKLADLAIQYNAIIVFEDLNMRFKQIRGGIEKSVYQQLEKALIDKLTF
KU516160 948 KDLKKGYISQVVRKLADLAIKHNAIIILEDLNMRFKQVRGGIEKSIYQQLEKALIDKLSF
******************** **** ********* ******* ************ *
SEQ ID 4 1005 LVEKEEKDVEKAGHLLKAYQLAAPFETFQKMGKQTGIVFYTQAAYTSRIDPVTGWRPHLY
KU516160 1008 LVDKGEKNPEQAGHLLKAYQLSAPFETFQKMGKQTGIIFYTQASYTSKSDPVTGWRPHLY
** * ** * ********** *************** ***** *** ***********
SEQ ID 4 1065 LKYSSAEKAKADLLKFKKIKFVDGRFEFTYDIKSFREQKEHPKATVWTVCSCVERFRWNR
KU516160 1068 LKYFSAKKAKDDIAKFTKIEFVNDRFELTYDIKDFQQAKEYPNKTVWKVCSNVERFRWDK
*** ** *** * ** ** ** *** ***** * ** * *** *** ******
SEQ ID 4 1125 YLNSNKGGYDHYSDVTKFLVELFQEYGIDFERGDIVGQIEVLETKGNEKFFKNFVFFFNL
KU516160 1128 NLNQNKGGYTHYTNITENIQELFTKYGIDITK-DLLTQISTIDEKQNTSFFRDFIFYFNL
** ***** ** * *** **** * ** * * ** * * ***
SEQ ID 4 1185 ICQIRNTNASELAKKDGKDDFILSPVEPFFDSRNSEKFGEDLPKNGDDNGAFNIARKGLV
KU516160 1187 ICQIRNTDDSEIAKKNGKDDFILSPVEPFFDSRKDN--GNKLPENGDDNGAYNIARKGIV
******* ** *** ***************** * ** ******* ****** *
SEQ ID 4 1245 IMDKITKFADENGGCEKMKWGDLYVSNVEWDNFV
KU516160 1245 ILNKISQYSEKNENCEKMKWGDLYVSNIDWDNFV
* ** * ************* *****
Thus, the claimed invention as a whole is prima facie obvious before the earliest effective filing date in the absence of evidence to the contrary.
Claims 25-27, 29, 33-34, 36-37, and 39 are rejected under 35 U.S.C. 103 as being unpatentable over Koonin, Murugan and Couvin as applied above, and further in view of Tu (Tu et al., Nucleic Acids Res 45: 11295-304 (2017)).
Regarding claims 33-34, although Koonin does not specifically teach wherein the target sequence is a sequence of a human, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to program the gRNA in the Cas12 systems taught by the combination of Koonin, Murugan and an NCBI database to target a sequence for gene editing of a gene of any mammal of interest (e.g., in a mammalian cell), especially in view of the references cited therein.
Furthermore in view of Tu specifically, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing use a Cas12 system taught by Koonin, Murugan, Couvin and an NCBI database to target a human gene for editing because Tu teaches engineered Cas12 system using Cas12 gRNAs targeting human DNA sequences (e.g., DNMT1) (Fig. 3). One of ordinary skill in the art with the goal of editing the genome of a human or nonhuman primate cell would be motivated to use a Cas12a-related system because Tu teaches Cas12a nucleases have equivalent editing efficiency to Cas9 but may have better specificity (pg. 11300, right col., last para., to pg. 11300, right col., 1st para.), especially in human cells (pg. 11296, left col., 2nd para.).
Regarding claim 36, Tu teaches using a catalytically active Cas12 variant which has dsDNA nuclease activity (Fig. 1; pg. 11296, right col., last para., to pg. 11299, left col., 1st para.; Fig. 2-3).
Regarding claim 37, Tu teaches Cas12a proteins cleave at sites distal to the target sequence (Fig. 3).
Regarding claim 39, Tu teaches wherein the Cas12a protein comprises nickase activity, which is required to generate one of the staggered ends (pg. 11296, left col., 1st para.).
Thus, the claimed invention as a whole is prima facie obvious before the earliest effective filing date in the absence of evidence to the contrary.
Claims 25-27, 29, 33-34, and 38 are rejected under 35 U.S.C. 103 as being unpatentable over Koonin, Murugan and Couvin as applied above, and further in view of Liu (Liu et al., Nat Commun 8: 2095 (2017)).
Regarding claims 33-34, although Koonin does not specifically teach wherein the target sequence is a sequence of a human, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to program the gRNA in a Cas12 system taught by Koonin, Murugan and an NCBI database to target a sequence for gene editing in any mammal, especially in view of the references cited therein.
Furthermore in view of Liu specifically, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to use a Cas12 system taught by Koonin, Murugan and Couvin to target a human gene for editing because Liu teaches engineered Cas12 system using Cas12 gRNAs targeting human DNA sequences (e.g., DNMT1) (Fig. 1-4). One of ordinary skill in the art with the goal of editing the genome of a human or nonhuman primate cell would be motivated to use a Cas12 system because Liu teaches Cas12-based systems can overcome some issues with Cas9-based systems, which suffer from in vivo delivery inefficiencies and large sizes (pg. 2, left col., last para., to right col., 1st para.).
Regarding claim 38, Koonin does not specifically teach using a catalytically active Cas12 variant.
However Liu teaches a catalytically inactive Cas12a-based (dAsCpf1-D908A) gene expression tuning system for use in human cells that overcomes some issues with dCas9-based systems, which suffer from in vivo delivery inefficiencies and large sizes (pg. 2, left col., last para., to right col., 1st para.), such as for engineering customized human cell signaling circuits and studying human cell biology (Abstract). Liu teaches using a Cas12a fused to a transcription repressor domain (KRAB) or a synthetic transcriptional activator domain (VPR) to program target gene expression with the benefit of lower mismatch tolerance than Cas9 and is more suitable for targeting AT rich promoters (id.; pg. 3, left col., last para., to pg. 5, left col., Fig. 1-2).
It would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to program a Cas12 system taught by Koonin, Murugan, Couvin and an NCBI database to target a sequence for transcription regulation in a human cell using a catalytically inactive Cas12 variant. One of ordinary skill in the art with the goal of editing the genome of a human genomic molecule would be motivated to use such a Cas12 system because Liu teaches it has advantages over traditional Cas9 systems, e.g., in terms of size, specificity, and targetable sequences.
Thus, the claimed invention as a whole is prima facie obvious before the earliest effective filing date in the absence of evidence to the contrary.
Claims 25-27, 29, 35-37, and 39 are rejected under 35 U.S.C. 103 as being unpatentable over Koonin, Murugan and Couvin as applied above, and further in view of Swarts (Swarts et al., Mol Cell 66: 221-33 (2017)).
Regarding claims 35-37, although Koonin does not specifically teach wherein the target sequence is a sequence of a bacteria, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to program the gRNA in a Cas12 system taught by Koonin, Murugan, Couvin and an NCBI database to target a bacterial sequence in view of Swarts teaching using Cas12a systems to target DNA sequences from bacteria (e.g., found in Francisella cf. novicida), such as a catalytically active Cas12 protein which cleaves at a site distal to the target sequence as does a wild-type Cas12a (Table S1; pg. e2, 3rd para.; Fig. 1, 3).
Regarding claim 39, Koonin does not expressly teach engineering a Cas12 protein to have nickase activity; however, Swarts teaches a mutated Cas12a (R1218A) that has nickase activity (Fig. 5). It would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to select the Cas12 system having nickase activity to cleave only one strand of a target DNA sequence to engineer a more specific type of primer editing, e.g., a base substitution, based on other well characterized Cas12 proteins with high homology.
Thus, the claimed invention as a whole is prima facie obvious before the earliest effective filing date in the absence of evidence to the contrary.
Response to Arguments
Applicant's arguments filed Dec. 31, 2025 have been fully considered but not found persuasive regarding the current obviousness rejection.
Motivations to find new Type V Cas12 variants provided in the prior art include the desire for new gene-editing tools with different characteristics, such as, inter alia, a different PAM specificity to increase the range of targets compared to already characterized Cas proteins for use in creating new CRISPR/Cas engineering tools as taught by Murugan (pg. 10, para. 4-5). This same general logic could be applied to Cas9 or any other known Cas protein to choose as an anchor sequence in an in silico computational analysis and subsequent validation by DNA cleavage assay. The whole entirety of public availably, searchable prokaryotic genomic databases in the prior art available for the obviousness analysis and these contain SEQ ID NO: 4.
Applicant traverses the previous obviousness rejection by arguing that there is no reasonable expectation of success before the time of filing to arrive at an engineered Cas12 system wherein the Cas12 protein comprises a sequence having at least 90% identity to instant SEQ ID NO: 3, 4, 5, or 222 and forms a complex with a non-natural gRNA to target a DNA sequence. However as laid out above, the prior art teaches multiple Cas12 CRISPR/Cas systems (e.g., Cas12a-e) were already known and had been used to engineer sequence programmable induction of double-stranded DNA breaks (DSBs) with high efficiencies by forming complexes with synthetic gRNAs (Koonin at Fig. 1, 3; pg. 71, left col., last para., to pg. 73, right col., 2nd para.). Furthermore, Koonin teaches searching databases to identify new Cas12 variants using in silico software analysis of prokaryotic genomic sequences anchored by sequences of known Cas genes or Cas12 proteins.
The logic of the obviousness rejection above is the artisan possessed both the necessary methods and the publicly available databases possessed the necessary sequences to arrive at the instantly claimed Cas12 inventions with a reasonable expectation of success, given the motivation to find any such variants of known Cas12 proteins by sequence homology/phylogenetics. While it may have been time-consuming, arduous and/or improbable to arrive at exactly instant SEQ ID NO: 4, this just represents one of many obvious species in the opinion of this office action absent any secondary indicium of nonobviousness.
The prior art shows repeated validation of new Cas proteins in the years following the advent of Streptomyces pyogenes Cas9 technologies, including Class II, Type V Cas12 proteins: Cas12b-i (Murugan et al., Mol Cell 68(1): 15-25 (2017) at pg. 2, para. 4-5; Shmakov et al., Mol Cell 60: 385-97 (2015); Burnstein2 (Burnstein et al., Nature 542: 237-41 (2017); Yan et al., Science 363: 88-91 (2019) at abstract). Thus, it is clearly within the skills of the ordinary artisan before the filing date to scientifically determine the functionality of a given new Cas protein identified via computational tools. This does not require any knowledge or consideration as to predicting protein folding as argued. Further, it is not clear Applicant performed any protein folding predictions to arrive at the claimed invention raising doubt to how enabling is the instant disclosure. The logic of the rejection does not require every computational identify Cas12 homolog to be functional but rather the ability to identify functional ones amongst candidates, e.g., using screening methods known in the prior art. If any of the present variants were identified by the prior art methods, then reconstituting the putative Cas9 protein with a synthetic cognate gRNA and performing an in vitro DNA cleavage assay is within the skills of the ordinary artisan in view this has been repeatedly performed on at least 30 different Cas proteins before the earliest effective filing date, including of diverse families and types as noted above, e.g., among Cas3 (Class 1, Type I), Cas 9 (Class 2, Type II), Cas13 (Class 2, Type VI), and Cas 12 Cas14 (Class 2, Type V). Similarly, the prior art provided tools to either experimentally or computationally identifying gRNA scaffolds and PAM specificities for newly discovered Cas proteins having expected functions (see e.g., Mendoza and Trinh, Biotechnol J 13: e1700595 (2018) at pg. 1, right col.; abstract).
Applicant argues that at the earliest effective timing of filing, Cas genes were challenging to identify and characterize, such as for Cas homologs having more divergent sequences more distantly related to already characterized ones, citing Koonin at pg. 2 and 6. Applicant’s response also argues the relied upon prior art does not teach how to adapt their method specific to other Cas homologs to the instantly claimed proteins of the Cas12 clade or its subtypes.
This is not found persuasive in view of the evidence. Each of Cas12b-i were novel and distinct subtypes upon their discovery using prior art methods known prior to the instant earliest priority date. Further, the prior art teaches conservation of portions of the nuclease domain and/or merely genomic position near easily identifiable CRISPR repeat arrays can be used using prior art methods to identify novel Cas proteins, regardless of low sequence homology conservation. For example, CjCas9 and FnCas9 were discovered using CRISPR locus sequence conservation despite these proteins having less than 40% sequence identity overall to other known Cas9 proteins (see e.g., Chylinski et al., RNA Biol 10: 726-37 (2013)).
Applicant traverse the previous rejection in part by arguing impermissible hindsight bias. In response to Applicant’s arguments, it must be recognized that any judgment of obviousness is in a sense a necessarily a reconstruction based on hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from Applicant’s disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392 (CCPA 1971).
The instant application makes the admission that the sequence of instant SEQ ID NO: 4 was identified in a publicly available NCBI database of metagenome sequences using CrisprCasFinder software, which could be filtered to only Class V Cas sequences (instant [0429]) and which was available at least by July 2018 in view of Couvin, among numerous other biological sequence searching and alignment tools. Without evidence to the contrary, the instant inventors only used conventional, well-known and/or routine methods (i.e., enabled prior art methods) to identify and test instant SEQ ID NO: 4 (as well as SEQ ID NOs: 3 and 5), which apparently have no unique feature over other Cas proteins in the prior art beyond being unique homologs produced by bacterial evolutionary history. The sheer volume of in silico data or subtype diversity for software analysis is not relevant in view of modern computing power and the ease of changing parameters and filters as of early 2019. For example, Applicant suggests in their remarks of July 3, 2025 (pg. 8) that a person of ordinary skill in the art before the instant filing date was already aware of groupings of Class 2 Type V CRISPR-Cas endonuclease systems based on bioinformatic analyses, e.g., based on sequence similarities, gene architecture, and other gene signature features allowing for Cas protein categorization and assignment to families of related Cas proteins, such as discussed at length in Koonin. Thus, methods were already known in the prior art (e.g., CrisprCasFinder) for finding, analyzing, and categorizing a given Cas12 sequence, such as a bioinformatically predictable Cas12 sequence present in the prior art databases, like instant SEQ ID NO:4.
While converting newly validated Cas nucleases into useful technological tools might require significant experimentation, merely expressing a predicted protein sequence and confirming actual target DNA cleavage as required by claim 25 was straightforward by Sept. 2019 in view of the dozens of Cas proteins being newly discovered since 2016. Although there is no motivation to specifically identify any single species of Cas12 variant (e.g., SEQ ID NO: 4) already present in publicly available bacterial metagenomic data before the earliest effective filing date, it is within the ordinary skills of the artisan to arrive at any Cas protein species having high sequence similarity to those known in the prior art and already available to the public before the date of the instant application. Absence evidence to the contrary, a Cas12 system comprising a Cas protein comprising at least one of instant SEQ ID NO: 4 is considered predictable to identify and validate using routine methods and lacking of any distinguishing features over Cas proteins already known in the prior art.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
Claims 25-26, 29-30, 36-37 and 39 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 11 of copending Application No. 17/772,960 (reference application).
Although the claims at issue are not identical, they are not patentably distinct from each other because claim 11 of the reference application discloses a CRISPR/Cas complex comprising a Cas12 protein (SEQ ID NO: 6) comprising instant SEQ ID NO: 4 and 222 and a guide RNA (gRNA), as shown below.
100.0% identity in 1281 residues overlap; Score: 6717.0; Gap frequency: 0.0%
Sequence4 1 MKKSIFDQFVNQYALSKTLRFELKPVGETGRMLEEAKVFAKDETIKKKYEATKPFFNKLH
Sequence6 1 MKKSIFDQFVNQYALSKTLRFELKPVGETGRMLEEAKVFAKDETIKKKYEATKPFFNKLH
************************************************************
Sequence4 61 REFVEEALNEVELAGLPEYFEIFKYWKRYKKKFEKDLQKKEKELRKSVVGFFNAQAKEWA
Sequence6 61 REFVEEALNEVELAGLPEYFEIFKYWKRYKKKFEKDLQKKEKELRKSVVGFFNAQAKEWA
************************************************************
Sequence4 121 KKYETLGVKKKDVGLLFEENVFAILKERYGNEEGSQIVDESTGKDVSIFDSWKGFTGYFI
Sequence6 121 KKYETLGVKKKDVGLLFEENVFAILKERYGNEEGSQIVDESTGKDVSIFDSWKGFTGYFI
************************************************************
Sequence4 181 KFQETRKNFYKDDGTATALATRIIDQNLKRFCDNLLIFESIRDKIDFSEVEQTMGNSIDK
Sequence6 181 KFQETRKNFYKDDGTATALATRIIDQNLKRFCDNLLIFESIRDKIDFSEVEQTMGNSIDK
************************************************************
Sequence4 241 VFSVIFYSSCLLQEGIDFYNCVLGGETLPNGEKRQGINELINLYRQKTSEKVPFLKLLDK
Sequence6 241 VFSVIFYSSCLLQEGIDFYNCVLGGETLPNGEKRQGINELINLYRQKTSEKVPFLKLLDK
************************************************************
Sequence4 301 QILSEKEKFMDEIENDEALLDTLKIFRKSAEEKTTLLKNIFGDFVMNQGKYDLAQIYISR
Sequence6 301 QILSEKEKFMDEIENDEALLDTLKIFRKSAEEKTTLLKNIFGDFVMNQGKYDLAQIYISR
************************************************************
Sequence4 361 ESLNTISRKWTSETDIFEDSLYEVLKKSKIVSASVKKKDGGYAFPEFIALIYVKSALEQI
Sequence6 361 ESLNTISRKWTSETDIFEDSLYEVLKKSKIVSASVKKKDGGYAFPEFIALIYVKSALEQI
************************************************************
Sequence4 421 PTEKFWKERYYKNIGDVLNKGFLNGKEGVWLQFLLIFDFEFNSLFEREIIDENGDKKVAG
Sequence6 421 PTEKFWKERYYKNIGDVLNKGFLNGKEGVWLQFLLIFDFEFNSLFEREIIDENGDKKVAG
************************************************************
Sequence4 481 YNLFAKGFDDLLNNFKYDQKAKVVIKDFADEVLHIYQMGKYFAIEKKRSWLADYDIDSFY
Sequence6 481 YNLFAKGFDDLLNNFKYDQKAKVVIKDFADEVLHIYQMGKYFAIEKKRSWLADYDIDSFY
************************************************************
Sequence4 541 TDPEKGYLKFYENAYEEIIQVYNKLRNYLTKKPYSEDKWKLNFENPTLADGWDKNKEADN
Sequence6 541 TDPEKGYLKFYENAYEEIIQVYNKLRNYLTKKPYSEDKWKLNFENPTLADGWDKNKEADN
************************************************************
Sequence4 601 STVILKKDGRYYLGLMARGRNKLFDDRNLPKILEGVENGKYEKVVYKYFPDQAKMFPKVC
Sequence6 601 STVILKKDGRYYLGLMARGRNKLFDDRNLPKILEGVENGKYEKVVYKYFPDQAKMFPKVC
************************************************************
Sequence4 661 FSTKGLEFFQPSEEVITIYKNSEFKKGYTFNVRSMQRLIDFYKDCLVRYEGWQCYDFRNL
Sequence6 661 FSTKGLEFFQPSEEVITIYKNSEFKKGYTFNVRSMQRLIDFYKDCLVRYEGWQCYDFRNL
************************************************************
Sequence4 721 RKTEDYRKNIEEFFSDVAMDGYKISFQDVSESYIKEKNQNGDLYLFEIKNKDWNEGANGK
Sequence6 721 RKTEDYRKNIEEFFSDVAMDGYKISFQDVSESYIKEKNQNGDLYLFEIKNKDWNEGANGK
************************************************************
Sequence4 781 KNLHTIYFESLFSADNIAMNFPVKLNGQAEIFYRPRTEGLEKERIITKKGNVLEKGDKAF
Sequence6 781 KNLHTIYFESLFSADNIAMNFPVKLNGQAEIFYRPRTEGLEKERIITKKGNVLEKGDKAF
************************************************************
Sequence4 841 HKRRYTENKVFFHVPITLNRTKKNPFQFNAKINDFLAKNSDINVIGVDRGEKQLAYFSVI
Sequence6 841 HKRRYTENKVFFHVPITLNRTKKNPFQFNAKINDFLAKNSDINVIGVDRGEKQLAYFSVI
************************************************************
Sequence4 901 SQRGKILDRGSLNVINGVNYAEKLEEKARGREQARKDWQQIEGIKDLKKGYISQVVRKLA
Sequence6 901 SQRGKILDRGSLNVINGVNYAEKLEEKARGREQARKDWQQIEGIKDLKKGYISQVVRKLA
************************************************************
Sequence4 961 DLAIQYNAIIVFEDLNMRFKQIRGGIEKSVYQQLEKALIDKLTFLVEKEEKDVEKAGHLL
Sequence6 961 DLAIQYNAIIVFEDLNMRFKQIRGGIEKSVYQQLEKALIDKLTFLVEKEEKDVEKAGHLL
************************************************************
Sequence4 1021 KAYQLAAPFETFQKMGKQTGIVFYTQAAYTSRIDPVTGWRPHLYLKYSSAEKAKADLLKF
Sequence6 1021 KAYQLAAPFETFQKMGKQTGIVFYTQAAYTSRIDPVTGWRPHLYLKYSSAEKAKADLLKF
************************************************************
Sequence4 1081 KKIKFVDGRFEFTYDIKSFREQKEHPKATVWTVCSCVERFRWNRYLNSNKGGYDHYSDVT
Sequence6 1081 KKIKFVDGRFEFTYDIKSFREQKEHPKATVWTVCSCVERFRWNRYLNSNKGGYDHYSDVT
************************************************************
Sequence4 1141 KFLVELFQEYGIDFERGDIVGQIEVLETKGNEKFFKNFVFFFNLICQIRNTNASELAKKD
Sequence6 1141 KFLVELFQEYGIDFERGDIVGQIEVLETKGNEKFFKNFVFFFNLICQIRNTNASELAKKD
************************************************************
Sequence4 1201 GKDDFILSPVEPFFDSRNSEKFGEDLPKNGDDNGAFNIARKGLVIMDKITKFADENGGCE
Sequence6 1201 GKDDFILSPVEPFFDSRNSEKFGEDLPKNGDDNGAFNIARKGLVIMDKITKFADENGGCE
************************************************************
Sequence4 1261 KMKWGDLYVSNVEWDNFVANK
Sequence6 1261 KMKWGDLYVSNVEWDNFVANK
*********************
Regarding instant claims 36-37 and 39, the CRISPR/Cas complex of reference claim 11 is implicitly and inherently catalytically active and also inherently cleaves (via a nickase activities) downstream (distal) of its target sequence as explained in a previous section due to the presence of instant SEQ ID NO: 4. This is a provisional nonstatutory double patenting rejection because the reference application claims have not in fact been patented.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ERIC J ROGERS whose telephone number is (571)272-8338. The examiner can normally be reached Monday - Friday 9:00-6:00.
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/ERIC J ROGERS/Examiner, Art Unit 1638
/KEVIN K HILL/Primary Examiner, Art Unit 1638
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