DETAILED ACTION
Status of the Application
Claims 1, 4-5, 7, 12-14, 16-17, 22-23, 47-49, 52-53, 76-88 are pending.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendment of claims 1,5, 7, 12-14, 16-17, 23, 47-49, 76, 79-80, addition of claims 81-88, cancellation of claim 11, and a declaration under 37 CFR § 1.132 by inventor Joseph Watts (hereinafter the Watts declaration), as submitted in a communication filed on 4/30/2026 is acknowledged.
As indicated in prior Office actions, Applicant elected Group 23 without traverse, which is drawn in part to an engineered Cas effector protein that comprises SEQ ID NO: 131, the heterologous polypeptide of SEQ ID NO: 1, and the combination of a wedge domain, a Rec1 domain, a Rec2 domain, a PAM interacting domain, a RuvC domain, a bridge helix and a Nuc domain, in a communication filed on 3/11/2024.
New claims 81-88 is directed in part to the elected invention. Claims 1 (linking), 4-5, 7, 12-14, 16-17, 22-23, 47-49, 52-53, 76-88 are at issue and will be examined to the extent they encompass the elected invention. Embodiments related to SEQ ID NO: 2-17, 125-130, 132, 157-168, 169-174 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 3/11/2024.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Claim Objections
Claim 12 is objected to due to the recitation of “two consecutive amino acids in the same Cas12a protein…wherein the two consecutive amino acids are selected from the group consisting of amino acid residues 290 and 291….and amino acid residues 295 and 295 of SEQ ID NO: 50”. Since the two consecutive amino acids are the amino acids of a Cas12a protein, the term should be amended to recited “two consecutive amino acids in the Cas12a protein of SEQ ID NO: 50…wherein the two consecutive amino acids are selected from the group consisting of amino acid residues 290 and 291….and amino acid residues 295 and 295 of the polypeptide of SEQ ID NO: 50”. Appropriate correction is required.
Claim 13 is objected to due to the recitation of “wherein the two consecutive amino acids are amino acid residues 291 and 292 of SEQ ID NO: 50”. To enhance clarity and to indicate that the amino acids belong to the Cas12a protein of SEQ ID NO: 50, the claim should be amended to recite “wherein the two consecutive amino acids are amino acid residues 291 and 292 of the polypeptide of SEQ ID NO: 50”. Appropriate correction is required.
Claim 48 is objected to due to the recitation of “…are two consecutive amino acids in the Cas12a protein and the two consecutive amino acids are amino acid residues 291 and 292 of SEQ ID NO: 50…”. To enhance clarity and to indicate that the amino acids belong to the Cas12a protein of SEQ ID NO: 50, the claim should be amended to recite “…are two consecutive amino acids in the Cas12a protein of SEQ ID NO: 50 and the two consecutive amino acids are amino acid residues 291 and 292 of the polypeptide of SEQ ID NO: SEQ ID NO: 50…”. Appropriate correction is required.
Claim 88 is objected to due to the recitation of “(a) an engineered Cas effector protein of claim 1….”. Since the Cas effector protein has been defined in claim 1, the term should be amended to recite “(a) the engineered Cas effector protein of claim 1….”. . Appropriate correction is required.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 7/7/2026, 5/28/2026, 4/27/2026, 3/25/2026 and 7/28/2026 are acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA )
Claims 1, 4-5, 7, 12-14, 16-17, 22-23, 47-49, 52-53, 76-80 remain rejected and new claims 81-86, 88 are 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. New grounds of rejection are necessitated by amendment.
Claim 1 (claims 4-5, 7, 12-14, 16-17, 22-23, 76-86, 88 dependent thereon) is indefinite in the recitation of “a first CRISPR-Cas effector polypeptide comprises…a first portion of a wedge domain (WED-1) and a Rec1 domain of a first Cas12a protein….second CRISPR-Cas effector polypeptide that comprises…aa Rec2 domain, a second portion of a wedge domain (WED-2), a PAM-interacting domain (PI), a third portion of a wedge domain (WED-3), a first portion of a RuvC domain (RuvC-1), a bridge helix domain, a second portion of a RuvC domain (RuvC-2), a Nuc domain, and a third portion of a RuvC domain (RuvC-3) of a second Cas12a protein, wherein the first portion, second portion, and third portion of the RuvC domain make up the complete RuvC domain of the second Cas12a protein…..wherein the heterologous polypeptide comprises an HNH domain wherein the HNH domain comprises a first nuclease domain and wherein the heterologous polypeptide is inserted between the Rec1 domain of the first CRISPR-Cas effector polypeptide and the Rec2 domain of the second CRISPR-Cas effector polypeptide…” for the following reasons.
The term “a first portion of a wedge domain (WED-1)….a second portion of a wedge domain (WED-2)….. a third portion of a wedge domain (WED-3),..” is unclear because one cannot determine if
the first CRISPR-Cas effector polypeptide comprises any fragment of a wedge domain (e.g., any fragment of 2 amino acids of a wedge domain) and the second CRISPR-Cas effector polypeptide comprises two fragments of any size of a wedge domain (e.g., any fragment of 2 amino acids of a wedge domain). In addition, it is unclear if the terms “WED-I”, “WED-2” and “WED-3” in parentheses are further
limiting the first, second and third portions of the wedge domain and how they are limiting the portions of the wedge domain. Furthermore, as written, it is unclear if the wedge domain of the first CRISPR-Cas effector polypeptide is the wedge domain of the same first Cas12a protein from which the Rec1 domain is from. Similarly, it is unclear if the wedge domain of the second CRISPR-Cas effector polypeptide is the wedge domain of the second Cas12a protein.
The term “a first portion of a RuvC domain (RuvC-1), … a second portion of a RuvC domain (RuvC-2), … and a third portion of a RuvC domain (RuvC-3) of a second Cas12a protein, wherein the first portion, second portion, and third portion of the RuvC domain make up the complete RuvC domain of the second Cas12a protein” is unclear because one cannot determine if the term refers to any fragment of any type of a RuvC domain. It is unclear if the terms “RuvC-1”, “RuvC-2”, and “RuvC-3” in parentheses are further limiting the first, second and third portions of a RuvC domain, and how they are limiting the portions of the RuvC domain. As known in the art, a Cas12a protein comprises a RuvC I domain, a RuvC II domain and a RuvC III domain which are not contiguous. Therefore, since there is no single RuvC domain, it is unclear as to what a complete RuvC domain is. As written, one cannot determine what is encompassed by the terms “a first portion of a RuvC domain”, “a second portion of a RuvC domain” and “a third portion of a RuvC domain”. Moreover, even if the three portions make up all of RuvC I, RuvC II, and RuvC III domains, it is unclear if, for example, the first portion of a RuvC domain can comprise all of a RuvC I domain and part of a RuvC II domain, the second portion can comprise only a fragment of a RuvC II domain, and the third portion can comprise a fragment of a RuvC II domain and all of a RuvC III domain. For examination purposes, it will be assumed that the first CRISPR-Cas effector polypeptide comprises a fragment of any wedge domain and a Rec1 domain of a first Cas12a protein, and the second CRISPR-Cas effector polypeptide comprises a Rec2 domain, a fragment of any wedge domain, a PAM-interacting domain, a fragment of any wedge domain, any fragment of any RuvC domain, a bridge helix domain, any fragment of any RuvC domain, a Nuc domain and any fragment of any RuvC domain of a second Cas12a protein. Correction is required.
Claim 47 (claims 48-49, 52-53, 82-83, 86 dependent thereon) is indefinite in the recitation of “..a first polypeptide that comprises a first portion of the wedge domain (WED-1) and a Rec1 domain of a Cas12a protein; a second polypeptide that comprises the Rec2 domain, as second portion of the wedge domain (WED-2), the PAM-interacting domain, a third portion of the wedge domain (WED-3), the RuvC domain, the bridge helix, and the Nuc domain of the Cas12a protein” for the following reasons.
The term “a first portion of a wedge domain (WED-1)….a second portion of a wedge domain (WED-2)….. a third portion of a wedge domain (WED-3),..” is unclear because one cannot determine if
the first polypeptide comprises any fragment of a wedge domain (e.g., any fragment of 2 amino acids of a wedge domain) and the second polypeptide comprises two fragments of any size of a wedge domain (e.g., any fragment of 2 amino acids of a wedge domain). In addition, it is unclear if the terms “WED-I”, “WED-2” and “WED-3” in parentheses are further limiting the first, second and third portions of the wedge domain and how they are limiting the portions of the wedge domain. Furthermore, the term “the RuvC domain” is unclear because as known in the art there is no single RuvC domain in a Cas12a protein. As indicated above, a Cas12a protein has a RuvC 1 domain, a RuvC II domain and a RuvC III domain. Therefore, one cannot determine which is “the” RuvC domain” being referred to. For examination purposes, it will be assumed that the engineered Cas effector protein comprises a first polypeptide having a fragment of any wedge domain and the Rec1 domain of a Cas12a protein, and the second polypeptide comprises the Rec2 domain, the PAM-interacting domain, the bridge helix, and the Nuc domain of the same Cas12a protein. Correction is required.
Claim 49 is indefinite in the recitation of “wherein the engineered Cas effector protein comprises the complete wedge domain of the Cas12a protein, wherein the wedge domain comprises the first portion of the wedge domain (WED-1), the second portion of the wedge domain (WESD-2), and the third portion of the wedge domain (WED-3)” for the following reasons. As known in the art, a Cas12a protein comprises three domains called WED I, WED II and WED III which are not contiguous. Therefore, since there is no single wedge domain, it is unclear as to what a complete wedge domain is. As written, one cannot determine what is encompassed by the terms “the first portion of a wedge domain”, “the second portion of a wedge domain” and “the third portion of a wedge domain”. Moreover, even if the three portions make up all of WED I, WED II and WED III domains, it is unclear if, for example, the first portion of a wedge domain can comprise all of a WED I domain and part of a WED II domain, the second portion can comprise only a fragment of the WED II domain, and the third portion can comprise a fragment of the WED II domain and all of a WED III domain. For examination purposes, it will be assumed that claim 49 is a duplicate of claim 47 as interpreted above. Correction is required.
Claim 76 is indefinite in the recitation of “wherein the RuvC domain of the second CRISPR-Cas effector polypeptide is inactivated by a D832A mutation corresponding to SEQ ID NO: 50, thereby reducing nickase activity on the nontarget strand of the engineered Cas effector protein” for the following reasons. Claim 1, from which claim 76 depends, refers to a complete RuvC domain. The RuvC domain of claim 76 is a variant of the RuvC domain of claim 1 as it contains an inactivating mutation. A mutant of the RuvC domain of claim 1 is not encompassed by the RuvC domain of claim 1 because the RuvC domain of claim 1 lacks the mutation. Therefore, the scope of claim 76 does not further limit the scope of claim 1 since the genus of the RuvC domains of claim 1 is not the genus of variants of RuvC domains of claim 76. In addition, it is unclear as to how a mutation (D832A) can correspond to an amino acid sequence (SEQ ID NO: 50). If the intended limitation is a substitution that corresponds to the substitution D832A in the polypeptide of SEQ ID NO: 50, the claim should be amended accordingly. For examination purposes, claim 76 will be interpreted as a duplicate of claim 1 as interpreted. Correction is required.
Claim 80 is indefinite in the recitation of “wherein the C-terminal amino acid residue of the first CRISPR-Cas effector polypeptide and the N-terminal amino acid residue of the second CRISPR-Cas effector polypeptide are two nonconsecutive amino acids in the same Cas12a protein and the heterologous polypeptide is between the two nonconsecutive amino acids, wherein the two nonconsecutive amino acids are selected from amino acid residues 270-305 of SEQ ID NO: 50, and wherein one of the two nonconsecutive amino acid residues is amino acid residue 283, 284, or 285 and the other of the two nonconsecutive amino acids residues is amino acid residue 293, 294, or 295” for the following reasons.
It is unclear as to how this limitation further limits the engineered Cas effector protein because if the C-terminal amino acid of the first CRISPR-Cas effector polypeptide and the N-terminal amino acid of the second CRISPR-Cas effector protein together do not form a dipeptide found in the “same” Cas12a protein, the dipeptide formed by these two amino acids can be any dipeptide. Moreover, the term “wherein the two nonconsecutive amino acids are selected from amino acid residues 270-305 of SEQ ID NO: 50” is unclear because one cannot determine if the claim is requiring that the N-terminal amino acid and the C-terminal amino acid be selected from any one of the amino acids in the fragment of amino acid residues 270-305, namely Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Val Leu Glu Val Phe Arg or Asn, and at the same time requiring that one of the amino acid residues be Arg, Glu, or Ser (amino acid residues 283-285 of SEQ ID NO: 50), and the other amino acid residue to be Tyr, Thr, or Ser (amino acid residues 293-295 of SEQ ID NO: 50). For examination purposes, it will be assumed that claim 80 is a duplicate of claim 7. Correction is required.
Claim 81 is indefinite in the recitation of “wherein the RuvC domain of the second Cas12a comprises an inactivating mutation, wherein the inactivation mutation is D832A corresponding to SEQ ID NO: 50” for the following reasons. Claim 1, from which claim 81 depends, refers to a complete RuvC domain. The RuvC domain of claim 81 is a variant of the RuvC domain of claim 1 as it contains an inactivating mutation. A mutant of the RuvC domain of claim 1 is not encompassed by the RuvC domain of claim 1 because the RuvC domain of claim 1 lacks the mutation. Therefore, the scope of claim 81 does not further limit the scope of claim 1 since the genus of the RuvC domains of claim 1 is not the genus of variants of RuvC domains of claim 81. In addition, it is unclear as to how a mutation (D832A) can correspond to an amino acid sequence (SEQ ID NO: 50). If the intended limitation is a substitution that corresponds to the substitution D832A in the polypeptide of SEQ ID NO: 50, the claim should be amended accordingly. For examination purposes, claim 81 will be interpreted as a duplicate of claim 1. Correction is required.
Claim 82 (claim 83 dependent thereon) is indefinite in the recitation of “wherein the RuvC domain of the second polypeptide comprises an inactivating mutation, wherein the inactivation mutation is D832A corresponding to SEQ ID NO: 50” for the following reasons. Claim 47, from which claim 82 depends, refers to a RuvC domain of a Cas12a protein. The RuvC domain of claim 82 is a variant of the RuvC domain of claim 47 as it contains an inactivating mutation. A mutant of the RuvC domain of claim 47 is not encompassed by the RuvC domain of claim 47 because the RuvC domain of claim 47 lacks the mutation. Therefore, the scope of claim 82 does not further limit the scope of claim 47 since the genus of the RuvC domains of claim 47 is not the genus of variants of RuvC domains of claim 82. In addition, it is unclear as to how a mutation (D832A) can correspond to an amino acid sequence (SEQ ID NO: 50). If the intended limitation is a substitution that corresponds to the substitution D832A in the polypeptide of SEQ ID NO: 50, the claim should be amended accordingly. For examination purposes, claim 82 will be interpreted as a duplicate of claim 47. Correction is required.
Claim 83 is indefinite in the recitation of “wherein the HNH domain is catalytically active and the RuvC is catalytically inactive, such that the engineered Cas effector protein is a target strand nickase” for the following reasons. Claim 47, from which claim 83 ultimately depends, refers to a RuvC domain of a Cas12a protein. The RuvC domain of claim 83 is an variant of the RuvC domain of claim 47 as it contains an inactivating mutation. The RuvC domain of claim 83 is not encompassed by the RuvC domain of claim 47 because the RuvC domain of claim 47 is not catalytically inactive. Therefore, the scope of claim 83 does not further limit the scope of claim 47 since the genus of the RuvC domains of claim 47 is not the genus of inactive variants of RuvC domains of claim 83. For examination purposes, claim 83 will be interpreted as a duplicate of claim 47. Correction is required.
When amending the claims, applicant is advised to carefully review all examined claims and make the necessary changes to ensure proper antecedent basis and dependency.
Claim Rejections - 35 USC § 112(a) or First Paragraph (pre-AIA )
Claims 16-17 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention. New grounds of rejection are necessitated by the introduction of new matter.
As set forth in MPEP 2163 (I)(B), new or amended claims which introduce elements or limitations that are not supported by the as-filed disclosure violate the written description requirement. See, e.g., In re Lukach, 442 F.2d 967, 169 USPQ 795 (CCPA 1971) (subgenus range was not supported by generic disclosure and specific example within the subgenus range); In re Smith, 458 F.2d 1389, 1395, 173 USPQ 679, 683 (CCPA 1972) (an adequate description of a genus may not support claims to a subgenus or species within the genus).
Claim 16 now requires the first CRISPR-Cas effector polypeptide to have an amino acid sequence at least 90% identical to amino acids 1-291 of SEQ ID NO: 50, and claim 17 now requires the second CRISPR-Cas effector polypeptide to comprise an amino acid sequence at least 90% identical to amino acids 292-1228 of SEQ ID NO: 50. While the specification provides support for variants of the polypeptide of SEQ ID NO: 50 having at least 90% sequence identity to the polypeptide of SEQ ID NO: 50, the Examiner has been unable to find support for a polypeptide having an amino acid sequence at least 90% identical to amino acids 1-291 of SEQ ID NO: 50 or a polypeptide having an amino acid sequence at least 90% identical to amino acids 292-1228 of SEQ ID NO: 50. There is no indication in the specification of (a) a first CRISPR-Cas effector polypeptide that comprises an amino acid sequence at least 90% identical to amino acids 1-291 of SEQ ID NO: 50, or (b) a second CRISPR-Cas effector polypeptide that comprises an amino acid sequence at least 90% identical to amino acids 292-1228 of SEQ ID NO: 50. Thus, there is no indication that an engineered Cas effector protein that comprises (i) a polypeptide having an amino acid sequence at least 90% identical to amino acids 1-291 of SEQ ID NO: 50, or (ii) a polypeptide having an amino acid sequence at least 90% identical to amino acid residues 292-1228 of SEQ ID NO: 50, was within the scope of the invention as conceived by Applicant at the time of the invention. Accordingly, Applicant is required to cancel the new matter in the response to this Office Action.
Claims 1, 4-5, 7, 12-14, 16-17, 22-23, 47-49, 52-53, 76-80 remain rejected and new claims 81-84, 86, 88 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention. This rejection as it relates to claim 80 is necessitated by amendment.
This rejection has been discussed at length in prior Office actions. It is maintained and further applied to new claims 81-84, 86, 88 for the reasons of record and those set forth below.
Applicant argues that the written description requirement does not require Applicant to disclose every species within the claimed genus. Applicant states that the specification discloses representative species, citing the proteins of SEQ ID NO: 125-132 which are LBCas12a-HNH fusion proteins. Applicant also refers to the proteins of SEQ ID NO: 2-17, 157-168 as comprising various combinations of Cas12a domains. Thus, Applicant is of the opinion that these disclosed species constitute disclosure of representative species of the claimed genus. Applicant states that the claims have been amended to include specific features tied to the disclosed sequences. Applicant states that independent claim 1 now recites specific subdomains, and that amended claims now require insertion sites, specific sequence identity thresholds and specific functional limitations. Applicant refers to the Watts declaration and states that in the declaration, it is indicated that Cas12a proteins have a conserved domain architecture, citing the teachings of Yamano et al. (Mol Cell 67:633-645, 2017; cited in the IDS). Applicant refers to the Watts declaration as indicating that Cas12a proteins from different bacterial species share the same conserved architecture, citing Yamano et al. Applicant states that one of skill in the art at the time of filing would have recognize that Cas12a proteins are defined by their conserved domain architecture rather than by primary sequence conservation. Applicant states that the specification provides examples of Cas12a proteins. Applicant refers to the working examples provided in the specification in support of the argument that Applicant possessed the claimed invention, citing Examples 1-4 as evidenced that Applicant conceived of and reduced to practice specific embodiments of the claimed genus.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection or avoid the rejection of claims 81-84, 86, 88. The Examiner acknowledges the amendments made to the claims as well as the Watts declaration, the teachings of the specification and the prior art. However, the Examiner disagrees with Applicant’s contention that the entire genus of proteins claimed is adequately described by the teachings of the specification and/or the prior art.
With regard to the arguments that (a) the disclosure provides representative species, such as the proteins of SEQ ID NO: 2-17, 125-132, and 157-168, and that these disclosed species constitute disclosure of representative species of the claimed genus, and (b) Examples 1-4 are evidence to show that Applicant conceived of and reduced to practice specific embodiments of the claimed genus, it is noted that while it is agreed that the specification provides working examples and disclose a few species of the claimed genus of proteins, all of the proteins of SEQ ID NO: 2-17, 125-132, and 157-168 are hybrid proteins that comprise the protein of SEQ ID NO: 1, which has been disclosed as a heterologous polypeptide that comprises a nuclease domain, wherein the protein of SEQ ID NO: 1 has been inserted at different positions within the polypeptide of SEQ ID NO: 50, which has been disclosed as a LbCas12a protein (from a Lachnospiraceae species). The species disclosed are all combinations of the same heterologous polypeptide having an HNH domain (SEQ ID NO: 1) being inserted at different locations within the same Cas12a protein (SEQ ID NO: 50).
With regard to the argument that the claims have been amended to include specific features tied to the disclosed sequences and that independent claim 1 now recites specific subdomains and that the amended claims include specific insertion sites, specific sequence identity thresholds and specific functional limitations, it is noted that while it is agreed that claims 1 and 47 requires certain domains and fragments thereof found in a Cas12a protein by name, neither claim 1 nor claim 47 recite any specific structural limitation regarding the domains or fragments thereof recited. Claims 1, 4-5, 7, 12-13, 47-49, 52-53, 76-83, 88 as currently presented require a genus of proteins that can comprise any amino acid sequence, thus having any structure. With regard to claim 14, only a small fraction of the entire claimed protein is structurally defined since the entire protein, based on the length of the protein of SEQ ID NO: 131, is approximately 1400 amino acids long and the limitation of claim 14 only defines at most 130 amino acids (130 = 0.9x144; SEQ ID NO: 1 has 144 amino acids ). With regard to claims 16-17, it is noted that either the second CRISPR-Cas effector protein or the first CRISPR-Cas effector protein can have any amino acid sequence, thus being structurally undefined. Moreover, in the case of claim 17, there is a significant amount of structural variability with respect to the second CRISPR-Cas effector protein. For example, SEQ ID NO: 50 has 1227 amino acids. Therefore, a protein having at least 90% sequence identity to amino acids 292-1228 of the polypeptide of SEQ ID NO: 50 can have up to 94 amino acid modifications (94 = 937x0.1; 937 = number of amino acids in the fragment of amino acids 292-1228 of SEQ ID NO: 50). With regard to claims 22-23, there is a significant amount of structural variability with respect to the members of the genus of proteins claimed. These claims encompass variants of the polypeptide of SEQ ID NO: 50 that can have up to 185 amino acid modifications (185 = 0.15x1227) when compared to the polypeptide of SEQ ID NO: 50, and variants of the polypeptide of SEQ ID NO: 131 that can have up to 69 amino acid modifications when compared to the polypeptide of SEQ ID NO: 131 (69 = 0.05x1377).
With regard to the argument that the Watts declaration indicates that Cas12a proteins have a conserved domain architecture, citing the teachings of Yamano et al. (Mol Cell 67:633-645, 2017; cited in the IDS), and that Cas12a proteins from different bacterial species share the same conserved architecture, it is noted that while it is agreed that Yamano et al. provides a generic diagram for the arrangement of domains in two bacterial Cpf1 proteins (Cas12a; Figure 2A) from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1), one cannot reasonably conclude that having this generic arrangement of domains can provide one of skill in the art enough information to allow one of skill in the art to envision the entire structure of any Cas12a protein. As known in the art, Cas12a (Cpf1) proteins are around 1300 amino acids long. Neither the specification nor the prior art disclose the motifs required in every one of the domains shown by Yamano et al. so that one could recognize whether a protein has those domains and could be classified as a Cas12a protein. As taught by Yamano et al., the degree of structural similarity among the two Cas12a proteins disclosed is low (page 633, right column, first full paragraph). Therefore, it is unclear as to whether or not one could use structural similarity to determine which proteins are Cas12a proteins. While Applicant argues that Cas12a proteins are defined by their conserved domain architecture rather than by primary sequence conservation, it is noted that one of skill in the art would require the amino acid sequence (structure) of a Cas12a protein to envision the structure of the hybrid protein claimed. Similarly, while the claims require any HNH domain that comprises a nuclease domain, the specification is silent with regard to the structural elements required in ay HNH domain that comprises a nuclease domain, including an HNH domain that comprises a nuclease domain that has target strand nickase activity.
The claims require a genus of Cas12a proteins having any structure and a genus of HNH domains that comprise a nuclease domain having any structure to combine them in the recited order and obtain a protein with the recited domains. While the specification discloses the protein of SEQ ID NO: 1 as an HNH domain that comprises a nuclease domain, the protein of SEQ ID NO: 50 as a Cas12a protein, and the prior art discloses a few Cas12a proteins, and a few Cas9 proteins having an HNH domain that comprises a nuclease domain, the specification and the prior art are silent with regard to the specific structural features required in any Cas12a protein and in any HNH domain having the recited nuclease domain. In addition, neither the specification nor the prior art discloses the structural elements within the polypeptide of SEQ ID NO: 1, the polypeptide of SEQ ID NO: 50 or the polypeptide of SEQ ID NO: 131 required in any variant having the recited % sequence identity so that the resulting variant has the same function as that of the polypeptide of SEQ ID NO: 1, the polypeptide of SEQ ID NO: 50 and the polypeptide of SEQ ID NO: 131, respectively. Neither the specification nor the prior art disclose the structural features of the Cas12a protein of SEQ ID NO: 50 required in any Cas12a protein. No disclosure of a structure/function correlation has been provided which would allow one of skill in the art to recognize which variants of the polypeptide of SEQ ID NO: 131 have the same nuclease activity as that of the polypeptide of SEQ ID NO: 131, or which proteins are Cas12a proteins, nucleases, target strand nickases, or non-target strand nickases.
As previously indicated, the total number of variants of the protein of SEQ ID NO: 131 having 95% sequence identity with the polypeptide of SEQ ID NO: 131 is 6.83x10205 variants. See calculations previously provided. It is reiterated herein that a sufficient written description of a genus of polypeptides may be achieved by a recitation of a representative number of polypeptides defined by their amino acid sequence or a recitation of structural features common to members of the genus, which features constitute a substantial portion of the genus. However, in the instant case, there is either no recited structural feature which is representative of all the members of the genus of engineered Cas effector proteins recited in the claims, or the recited structural feature, e.g., 95% sequence identity to SEQ ID NO: 131, is not representative of all the members of the genus of engineered Cas effectors recited since there is no information as to which are the structural elements within the polypeptide of SEQ ID NO: 131 that are essential for the desired activity, which are the remaining structural elements required in the recited polypeptides in addition to those recited in the claims such that the desired nuclease activity is displayed, or a correlation between structure and function which would provide those unknown structural features. While Applicant argues that the few species disclosed are representative of the structure of all the members of the genus, it is reiterated herein that the prior art, as evidenced by Witkowski et al., Seffernick et al. and Tang et al., teaches how even highly structurally homologous polypeptides can have different enzymatic activities. Due to the fact that the specification only discloses a limited number of species of the genus of engineered Cas effector proteins required by the claims, and the lack of description of any additional species by any relevant, identifying characteristics or properties, one of skill in the art cannot reasonably conclude that the entire genus of proteins claimed is adequately described by the teachings of the specification and/or the prior art.
Claims 1, 4-5, 7, 12-14, 16-17, 22-23, 47-49, 52-53, 76-80 remain rejected and new claims 81-84, 86, 88 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 the protein of SEQ ID NO: 131, does not reasonably provide enablement for (I) an engineered Cas effector protein having any structure and function, wherein said engineered Cas effector protein comprises (a) two polypeptides (i.e., two CRISPR-Cas effector proteins) wherein each of these two polypeptides comprise fragments or domains of a Cas12a protein having any structure , and (b) a heterologous polypeptide that comprises a nuclease domain having any structure, or (II) variants of the polypeptide of SEQ ID NO: 131 or SEQ ID NO: 50. 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. This rejection as it relates to claim 80 is necessitated by amendment.
This rejection has been discussed at length in prior Office actions. It is maintained and further applied to new claims 81-84, 86, 88 for the reasons of record and those set forth below.
Applicant argues that the pending claims as amended are commensurate with what the specification teaches a person of ordinary skill in the art to make and use without undue experimentation. Applicant states that the claims do not require any structure. Applicant states that the specification teaches and the claims reflect a defined Cas12a structure in which discrete domains and multi part domains occupy specific positions. Applicant states that the disclosure ties the claims to Cas12a architectures described in the literature and within the specification. Applicant states that the Watts declaration explains how the examples provided in the specification present a clear, structure-guided domain-based blueprint for practicing the invention without undue experimentation. Applicant states that the specification teaches HNH domains from non-CRISPR sources and provides specific sequences for HNH domains that are not from a CRISPR-Cas effector protein. Applicant submits that the specification further provides explicit sequence identity thresholds and further contemplates inactivating substitutions in HNH to control nickase activity. Applicant points out that the disclosure provides working examples enabling guidance for constructing and validating the claimed engineered proteins citing Example 1. Applicant states that Example 4 reports expression and purification of multiple engineered constructs and validates nickase activity. Applicant states that the specification discloses that engineered proteins may have specified levels of sequence identity to all or portions of identified wild-type CRISPR-Cas effectors. Applicant states that these disclosures bound permissible sequence variability in the claims and provide concrete examples from which a person of ordinary skill in the art can make variants without undue experimentation. Applicant refers to the Wands factors and state that the claims as amended are defined by domain architecture constrains, by enumerated domain portions, by explicit sequence identifiers and by functional nickase requirements. Applicant is of the opinion that the field of CRISPR engineering was mature as of filing, with well characterized Cas12a domain architecture and widely adopted design, expression and assay methodologies. Applicant states that one of skill in the art with experience in protein engineering and CRISPR systems could readily implement the claimed invention with the teachings of the specification. Applicant states that domain location and multi-part domain composition are conserved in Cas12a proteins and that implementing the disclosed domain order and placing the heterologous HNH at the disclosed Rec1/Rec2 interface yields predictable activity outcomes. Applicant states that the examples use routine expression systems, purification and nickase assays typical of the art. Applicant is of the opinion that the disclosure teaches where to position the heterologous domains, what sequences to use or vary, what domain architecture to maintain and how to assay function, thus providing sufficient guidance that obviates undue experimentation.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection or avoid the rejection of claims 81-84, 86, 88. The Examiner acknowledges the amendments made to the claims as well as the Watts declaration, the teachings of the specification and the prior art. However, the Examiner disagrees with Applicant’s contention that the entire scope of the claims is fully enabled by the teachings of the specification and/or the prior art.
With regard to the arguments that (a) the specification teaches and the claims reflect a defined Cas12a structure in which discrete domains and multi part domains occupy specific positions, and (b) the disclosure ties the claims to Cas12a architectures described in the literature and within the specification, it is noted that as set forth in MPEP § 2111.01 (II), one cannot import into a claim limitations from the specification. Applicant is reminded that the claims are not limited to the proteins specifically disclosed in the specification by amino acid sequence. As indicated above, claims 1, 4-5, 7, 12-13, 47-49, 52-53, 76-83, 88 as currently presented require a genus of proteins that can comprise any amino acid sequence, thus having any structure. With regard to claim 14, only a small fraction of the entire claimed protein is structurally defined since the entire protein, based on the length of the protein of SEQ ID NO: 131, is approximately 1400 amino acids long and the limitation of claim 14 only defines at most 130 amino acids. With regard to claims 16-17, it is noted that either the second CRISPR-Cas effector protein or the first CRISPR-Cas effector protein can have any amino acid sequence, thus being structurally undefined. Moreover, in the case of claim 17, there is a significant amount of structural variability with respect to the second CRISPR-Cas effector protein. With regard to claims 22-23, there is a significant amount of structural variability with respect to the members of the genus of proteins claimed. These claims encompass variants of the polypeptide of SEQ ID NO: 50 that can have up to 185 amino acid modifications when compared to the polypeptide of SEQ ID NO: 50, and variants of the polypeptide of SEQ ID NO: 131 that can have up to 69 amino acid modifications when compared to the polypeptide of SEQ ID NO: 131.
With regard to the argument that (i) the Watts declaration explains how the examples provided in the specification present a clear, structure-guided domain-based blueprint for practicing the invention without undue experimentation, and (ii) the specification teaches HNH domains from non-CRISPR sources and provides specific sequences for HNH domains that are not from a CRISPR-Cas effector protein, it is reiterated herein that to practice the entire scope of the claimed invention, one of skill in the art would require the structure of any Cas12a protein and the structure of any HNH domain that comprises a nuclease domain so that when combined it can display the desired enzymatic activity. While the prior art discloses a limited number of Cas12a proteins and a limited number of Cas9 proteins having an HNH domain that comprises a nuclease domain so that they can be used to make a fusion protein as claimed, neither the specification nor the prior art disclose the structural features required in any Cas12a protein or HNH domain as required by the claims. Please note that the claims are not limited to hybrid proteins that can be obtained solely by using the Cas12a protein of SEQ ID NO: 50 and the nuclease domain of SEQ ID NO: 1.
With regard to the argument that the specification (i) further provides explicit sequence identity thresholds and further contemplates inactivating substitutions in HNH to control nickase activity, (ii) provides working examples including Example 1, (iii) discloses how to make multiple engineered constructs and validates nickase activity, (iv) discloses that engineered proteins may have specified levels of sequence identity to all or portions of identified wild-type CRISPR-Cas effectors, and (v) teaches where to position the heterologous domains, what sequences to use or vary, what domain architecture to maintain and how to assay function, it is noted that the issue in the instant case is not whether the specification and/or the prior art provides enzymatic assays, purification protocols or methods to make a recombinant protein once the amino acid sequence of the desired protein is known. Instead the issue in the instant case is whether the specification and/or the prior art provides the structural features of any Cas12a protein and any HNH domain that comprises a nuclease domain so that they can be combined to obtain proteins as claimed with the disclosed enzymatic activity. It is reiterated herein that the amino acid structure of the Cas12a proteins and HNH domains recited is essential to make the claimed proteins. Moreover, while Applicant states that the specification provides explicit sequence identity thresholds and discloses that engineered proteins may have specified levels of sequence identity to all or portions of identified wild-type CRISPR-Cas effectors, it is noted that the specification fails to disclose which is the level of identity with the polypeptide of SEQ ID NO: 50, SEQ ID NO: 1, or SEQ ID NO: 131 required to maintain the required activity. There is no disclosure of how much structural variability is found among Cas12a proteins with regard to the polypeptide of SEQ ID NO: 50, or how much structural variability is found among HNH domains having a nuclease domain that can be used to make the claimed proteins with respect to the nuclease domain of SEQ ID NO: 1.
With regard to arguments related to the Wands factors indicating that (i) the field of CRISPR engineering was mature as of filing, with well characterized Cas12a domain architecture and widely adopted design, expression and assay methodologies, (ii) one of skill in the art with experience in protein engineering and CRISPR systems could readily implement the claimed invention with the teachings of the specification, and (iii) domain location and multi-part domain composition are conserved in Cas12a proteins and that implementing the disclosed domain order and placing the heterologous HNH at the disclosed Rec1/Rec2 interface yields predictable activity outcomes, it is noted that the field of CRISPR engineering is not mature as claimed as evidenced by the fact that more types of CRISPR proteins and new CRISPR proteins have been discovered and are being discovered since the first Cas9 and Cpf1 proteins were initially described. While it is agreed that a generic Cas12a domain architecture has been described, it is noted that to readily implement the claimed invention, one of skill in the art would require the actual amino acid sequences of the Cas12a proteins and HNM domains recited in the claims.
It is reiterated herein that the specification discloses the amino acid sequence of a limited number of engineered Cas effector proteins, including the protein of SEQ ID NO: 131, and a limited number of Cas12a proteins, such as the protein of SEQ ID NO: 50, and a limited number of nuclease domains, such as the protein of SEQ ID NO: 1, as working examples. Also, the art discloses a limited number of Cas12a proteins, a limited number of nucleases, a limited number of target strand nickases and non-target strand nickases, and a limited number of proteins that can bind to a guide RNA. However, neither the specification nor the art provide a correlation between structure and function such that one of skill in the art can envision the structure of any Cas12a protein, any HNH domain that comprises a nuclease domain, any nickase or any protein that can bind to a guide RNA. In addition, the art does not provide any teaching or guidance as to which changes can be made to the protein of SEQ ID NO: 131 such that the resulting variant would display the desired functional characteristics, or the general tolerance of nucleases to structural modifications and the extent of such tolerance. It is reiterated herein that there is a high level of unpredictability associated with accurate functional annotation of proteins based solely on structural homology, and that there is a high level of unpredictability with regard to the modification of a protein’s amino acid sequence to obtain the desired activity without any guidance/knowledge as to which amino acids in a protein are tolerant of modification and which ones are conserved. See teachings of Singh et al. and Sadowski et al. previously introduced.
At the time of the invention, it was not routine in the art to screen by a trial and error process for an essentially infinite number of proteins to find (i) Cas12a proteins, (ii) HNH domains comprising nuclease domains, (iii) target strand nickases or non-target strand nickases, or (iv) polypeptides having a nuclease domain that can be combined with any portion of any Cas12a protein and can have nuclease activity. In the absence of (a) a rational and predictable scheme for selecting those proteins most likely to have the desired functional features, and/or (b) a correlation between structure and function, one of skill in the art would have to test an essentially infinite number of proteins to determine which ones have the desired functional characteristics. While it is agreed that a considerable amount of experimentation is permissible if it is merely routine, in the instant case, testing an infinite number of proteins to find those having the desired function without a reasonable amount of guidance with respect to the direction in which the experimentation should process is not deemed routine experimentation. Therefore, contrary to Applicant’s assertions, neither the specification nor the prior art provides enablement to the full scope of the claimed invention.
Allowable Subject Matter
The subject matter of claim 85, which is directed to the protein of SEQ ID NO: 131, appears to be allowable over the prior art of record.
Claim 87 is allowable over the prior art of record.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
Applicant is advised that any Internet email communication by the Examiner has to be authorized by Applicant in written form. See MPEP § 502.03 (II). Without a written authorization by Applicant in place, the USPTO will not respond via Internet email to any Internet correspondence which contains information subject to the confidentiality requirement as set forth in 35 U.S.C. 122. Sample written authorization language can be found in MPEP § 502.03 (II). An Authorization for Internet Communications in a Patent Application or Request to Withdraw Authorization for Internet Communications form (SB/439) can be found at https://www.uspto.gov/patent/forms/ forms-patent-applications-filed-or-after-september-16-2012, which can be electronically filed.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to DELIA M RAMIREZ, Ph.D., whose telephone number is (571) 272-0938. The examiner can normally be reached on Monday-Friday from 8:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert B. Mondesi, can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
/DELIA M RAMIREZ/Primary Examiner, Art Unit 1652
DR
July 28, 2026
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