DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant's election with traverse of Group I, which reads on claims 1-2, 8-14, and 19-22 and the Species election of SEQ ID NOs 1 and 15, where the complex is drawn to the nickase itself, in the reply filed on 5/5/2026 is acknowledged. The traversal is on the ground(s) that searching one group would overlap with the other groups. This argument is not found to be persuasive because each of the invention groups would require separate, unique considerations and searches, for instance, for each embodiment of the method claims and the different cell types comprised by the method claims of Groups II and the cell types of Groups III-IV.
Upon searching Applicant’s elected species of SEQ ID NO: 15, this specific sequence was found to be free of the art. Thus, the search was expanded to the broader limitations recited in claim 1.
The requirement is still deemed proper and is therefore made FINAL.
Application Status
This action is written in response to applicant’s correspondence received on 7/3/2023. Claims 1-2, 8-14, 19-22, 26-27, 38-40, 46, 49-58, 65-67, 73, 76-85, 88, and 92-95 are pending. Claims 26-27, 38-40, 46, 49-58, 65-67, 73, 76-85, 88, and 92-95 are withdrawn from consideration as they are drawn to non-elected subject matter. Claims 1-2, 8-14, and 19-22 are currently under examination.
Claim Rejections - 35 USC § 112
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.
Claims 1-2, 8-14, and 19-22 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 1, claim 1 recites an engineered Cas12a with nickase activity, where furthermore the Cas12a comprises a combination of mutations within the lid domain, where the lid domain is in reference to SEQ ID NO: 13. SEQ ID NO: 13 is produced below:
LXXGFKXXRXXXXEXXX (SEQ ID NO: 13)
As shown above, SEQ ID NO: 13 comprises six defined amino acid residues (bold and underlined, above). The remaining residues recited are variable “X” residues. Claim 1 is unclear because it is unclear what constitutes a mutation in SEQ ID NO: 13. It is unclear if defining an “X” residue constitutes a mutation, or if a “mutation” is only in reference to one of the six defined residues.
Claims 2, 8-14, and 19-22 depend from claim 1 and do not resolve this 112(b) issue and are therefore also rejected.
Claim Interpretation
For the purposes of the art rejections which follow, claim 1 is being interpreted to mean that a mutation in SEQ ID NO: 13 means that an “X” in SEQ ID NO: 13 has been defined/mutated.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-2, 8-14, and 19-22 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.
MPEP 2163.II.A.3.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by 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 function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Regents of the University of California v. Eli Lilly & Co, 119 F.3d at 1568, 43 USPQ2d at 1406.
Regarding claim 1, claim 1 is drawn to an engineered Cas12a nuclease or catalytically active fragment therefore, where the Cas12a nuclease comprises mutations in the lid domain, and furthermore comprises a core lid domain structure of SEQ ID NO: 13. Regarding claim 1, three separate 112(a) issues exist for the claim language of claim 1.
Issue 1
Claim 1 broadly encompasses “fragments” of a Cas12a nuclease, which are recited with the functionality of having broad spectrum nickase activity. This claim language is problematic because the genus of “fragments” of Cas12a were not characterized in the specification, which broadly includes any portion of a Cas12a which simply comprise SEQ ID NO: 13, including a peptide comprising solely SEQ ID NO: 13. The Applicant did not perform sufficient fragmentation analysis of the recited Cas12a nickases to show possession of the genus of “catalytically active fragment” which would be functionally capable of acting as a nickase. Furthermore, as discussed further below, it is known in the art that such truncations or fragmentation of a Cas12a do not predictably act as functional nuclease/nickases (see discussion on state of the art, below).
Issue 2
Furthermore, claim 1 defines Cas12a nickase activity by the presence of a mutated lid domain (SEQ ID NO: 13, and mutations within the lid domain). Claim 1 therefore defines a Cas12a protein by its comprising SEQ ID NO: 13. This claim language is problematic because it is known in the art that the “lid” domain (i.e., SEQ ID NO: 13) is present in undefined proteins, where the status of such undefined proteins as Cas12a is unknown (see discussion, below). As such, the mere presence of SEQ ID NO: 13 is not sufficient to functionally define a protein as being a Cas12a (see below). Thus, the Applicant was not in possession of the genus of Cas12a enzymes which are defined by the presence of SEQ ID NO: 13.
Issue 3
In addition, the Cas12a enzymes are recited with mutations, including embodiments such as mutations in the lid domain comprising a single point mutation (step “iv” of claim 1) or including three consecutive point mutations (step “i” of claim 1). This claim language is problematic because it requires functional limitations: the Cas12a enzyme with a mutated lid domain must function as a nickase. However, as discussed further below, the specification demonstrates that certain variants of Cas12a with point mutations have wide variability with regards to their ability to function as nickases. The genus of “mutations in the lid domain” which render Cas12a variants with nickase functionality was therefore not shown to be in possession by the Applicant, as the specification shows variability in whether or not such mutations will yield Cas12a nickases. Furthermore, the Applicant does not provide a way to predict which lid domain mutations will yield Cas12a nickases; a structure/function relationship is therefore not established by the specification and the Applicant has not shown possession of the claimed genus of mutated lid domains. Finally, the state of the art demonstrates that the lid domain is critical for the functionality of the protein, thus supporting the fact that the genus of “mutations” in the lid domain which would yield functional nickases is unpredictable (see state of the art discussion, below).
Guidance in the Specification
With regards to the specification, the Applicant has offered Examples 1-11. Example 1 describes a rational protein design overview of LbCas12a and FnCas12a. Example 2 describes in silico identification of core structures of Cas12a proteins using bioinformatic alignment algorithm approaches, where the lid domain is said to be conserved across known and unknown Cas12a proteins (paragraph 211 of specification). Furthermore, Example 2 recites that the lid domain varies in length according to a given Cas12a (see Figure 1, reproduced in part, below):
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The lid domain structure is in bold in Figure 1 (reproduced above), and SEQ ID NO: 13 (the lid domain, per claim 1) sequence is given below:
LXXGFKXXRXXXXEXXX (SEQ ID NO: 13)
As seen above, the “R” position of SEQ ID NO: 13 is followed by four “X” residues (i.e., any residue) and then an “E” residue. As seen in Figure 1, only the AsCas12a protein (SEQ ID NO: 2, top row) in fact comprises the lid domain motif presented in SEQ ID NO: 13, as each of the other Cas12a proteins only have three residues following the defined “R” residue. Thus, at face value, SEQ ID NO: 13 is not a defining motif for a lid domain, as the Applicant has shown that Cas12a enzymes SEQ ID NOs 3-12 comprise different lid domains compared with SEQ ID NO: 13 by one residue. Thus, the specification and Figure 1 speak to uncertainty in defining a Cas12a protein by using SEQ ID NO: 13, as the Applicant has shown that SEQ ID NO: 13 is not fully required to be present to define a Cas12a protein. Thus, SEQ ID NO: 13 is not “a suitable consensus motif applicable for all Cas12a enzymes described and yet to be described” as stated in paragraph 211 of the specification because the SEQ ID NO: 13 motif is not found in SEQ ID NOs 2-12 (Figure 1).
Regarding Example 3 describes a fluorescence-based in vivo screening assay for nickase activity of select mutations in Cas12a variants.
Example 4 describes further semi-random mutational analysis where the lid domain was targeted for mutagenesis analysis and subjected to the fluorescence assay/screening technique described in Example 3. The RuvCL-del1 variant of Cas12a (SEQ ID NO: 15) is generated and shows high nickase activity. Variants with enhanced nickase activity are identified (Figure 5E).
Example 5 recites that a cell-free system (TXTL) was used in which the genes for a Cas12a variant, a guide RNA, and GFP are coexpressed in the same well. Inhibition of GFP fluorescence indicates cleavage activity. This system was adapted as a double-nicking assay in vitro, using guide RNA pairs to create a DSB using two independent nicks, and was validated with Cas9 O10A as a control.
In Example 6, a method is described where the selected variants of Cas12a nickase are introduced in Bacillus subti!is to evaluate its activity in vivo. Specific plasmids were constructed for gene deletion and integration using a Gibson assembly-based strategy and kanamycin selection, allowing us to distinguish between nickase activity and nuclease activity.
Example 7 describes the use of the Cas12a variants in plants, while Example 8 describes gene editing in Ashbya gossypii (fungus). Examples 9 and 10 describe nickase activity of select Cas12a nickases in yeast and mammalian cells, respectively.
In Example 11, the Applicant indicates that the selected nickase variants are planned to be evaluated in base editing systems (with cytidine and/or adenosine
deaminases) and prime editing systems (with different reverse transcriptases and pegRNA designs) in cultured plants and, optionally, in fungal and human cells. Preliminary results in rice protoplasts showed that the nickase variants reduce base editing efficiency by cutting the edited strand, but a strategy inspired by the Cas9 PE3
system is proposed to reverse this effect
The specification demonstrates that not all mutations within the lid domain produce nickases with similar degrees of functionality. For instance, in Example 4, pRV26180 comprises three consecutive mutations (“KVE” is mutated from the WT to “SSS” and “SR” is mutated from the wildtype to “FL,” see Figure 5A), where such mutations produce only limited nickase functionality (see Figure 5B, where the fluorescence of pRV26180 is comparable to the wildtype LbCas12a). Thus, mutations with at least 3 consecutive point mutations in the lid domain do not always render Cas12a enzymes with similar nickase functionality. The Applicant does not recite or teach a way to predict which lid domain mutations will render functional nickases, and only offers examples of confirmed nickases identified by positive mutational analysis screening using a fluorescent reported, where Cas12a variants which were not functional as nickases were not evaluated. The Applicant has therefore inherently described and shown possession of mutants which they have tested and sequenced. Given the variability in the ability of these mutants to function as nickases, it is unpredictable if all point mutations/triple mutations/deletions will render functional nickase Cas12a enzymes. For instance, the Applicant shows results for lid domain mutations in Figure 4, where lid domain mutations do not necessarily yield nickase functionality when point mutations are present (see Figure 4 and paragraph 220). The specification therefore does not characterize the genus of “mutant lid domain” which render nickase functionality commensurate in scope with what is claimed because the specification itself demonstrates that not all mutants in the lid domain yield nickase activity, nor does the Applicant offer a way to reliably forecast what specific combination of mutations will yield a functional Cas12a nickase.
State of The Art
Issue 1 – Fragments of Cas12a Enzymes do not function predictably
Regarding the state of the art, it is known that Cas12a enzymes use multiple domains to exact their enzymatic function. For example, Strohkendl (Strohkendl et al. Mol Cell. 2024 Jul 25;84(14):2717-2731.e6) is a research article that focuses on domains and functionality of Cas12a (Title, Abstract, and throughout). Strohkendl teaches that:
“it is known that the Rec lobe must rearrange to accommodate the helical 20-bp R-loop and expose the RuvC nuclease domain. Single-molecule studies have further described protein conformational changes that enable double-stranded DNA cleavage. Given the importance rate-limiting R-loop formation has in nuclease activation and off-target rejection, structures of the R-loop during formation would lead to better understanding of the underlying mechanism driving Cas12a activity and specificity. Additionally, as a new wave of minimal type V CRISPR effectors are being pursued for genome editing applications, understanding the functional importance of lost domains could guide engineering efforts to improve their efficiency,” (page 2717, right column, first paragraph).
Thus, Strohkendl teaches that Cas12a structures comprise complex architecture which spans multiple domains required for functionality, where furthermore additional understanding of R-loop structure is required to understand underlying mechanisms of Cas12a activity (above). Furthermore, while Strohkendl teaches that minimal effector sizes of CRISPR effectors are being investigated, there is still further understanding required to understand the “functional importance of lost domains” which could guide engineering efforts. Strohkendl therefore teaches that engineering CRISPR effectors such as Cas12a with lost domains (i.e., “fragments” of Cas12a) is therefore not a fully characterized or developed field, where further investigation is required in order to arrive at, engineer, and understand the functional importance of fragmented Cas12a enzymes. Given that the Applicant has not characterized “fragments” of Cas12a enzymes and/or truncated forms of such proteins, and that it is known in the art that such Cas12a proteins require complex protein architecture, where the functional relevance of “lost domains” (i.e., fragments) is still being characterized, the Applicant has not shown possession of the genus of “fragments” of the recited Cas12a enzymes as the art teaches unpredictability and complexity of such a genus. The Applicant has not characterized a minimal Cas12a fragment which would function as a nickase nor given sufficient guidance to show possession of such fragments.
Issue 2 – The presence of SEQ ID NO: 13 Does Not Predict that a Protein is a Cas12a enzyme
It is known in the art that the mere presence of SEQ ID NO: 13 in a protein sequence does not render Cas12a functionality onto the protein. For instance, BLAST database searches revealed the hypothetical protein taught by NCBI BLAST NJK71813.1 (hypothetical protein HC932_06315, Thermales Bacterium, NCBI BLAST search results, Accession number NJK71813.1, published 4/5/2020, hereinafter NJK). An alignment of the lid domain/SEQ ID NO: 13 is shown below with residues 78-96 of NJK:
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409
1568
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As seen above, NJK is an undefined, hypothetical protein which also comprises the “lid” domain found in SEQ ID NO: 13. Furthermore, NJK is only 237 amino acids long (see page 2 of NJK), while the Cas12a variants identified in the instant specification (e.g., SEQ ID NOs 1-12), range in sizes between 1206 residues (instant SEQ ID NO: 9) and 1373 residues (SEQ ID NO: 8). Thus, NJK, which is only 237 residues in length is unlikely to function as a Cas12a enzyme, as the recited/known Cas12a enzymes are at least roughly 1200 residues long. Thus, the genus of enzymes which are functionally defined as Cas12a enzymes by the presence of SEQ ID NO: 13 was not demonstrated to be in possession by the Applicant because it was known in the art that hypothetical, undefined proteins with significantly shorter lengths such as NJK are also known to comprise SEQ ID NO: 13, where furthermore given the significant differences in length NJK is unlikely to function as a Cas12a enzyme.
Issue 3 – Lid Domain Mutations
Regarding issue 3, given the relatively limited knowledge in the state of the art surrounding the outcomes of mutating the lid domain and its effects on nickase activities of Cas12a, a higher burden is placed on the Applicant to show possession of the genus of “mutated” lid domain which confers nickase activity. The specification demonstrates that point mutations in the lid domain do not necessarily yield nicikase Cas12a enzymes (e.g., Figure 4, paragraph 220). Without mechanistic or structure/functional explanations for the variability in the results, the Applicant has not shown possession of the broad genus of mutants presently recited in claim 1.
In addition, Stella (WO 2019/233990, A1) is a patent document which teaches alterations in Cas12a proteins, specifically in the lid domain (Title, Abstract, claims, throughout). Stella teaches that:
“A Cpf1 mutant carrying a substitution of the Lid domain (SEQ ID NO: 38) is NOT able to cut the target-strand and the non-target-strand when they are in the double strand DNA form. The mutant is not able to cut the target-strand alone as single strand DNA (Figure 4A, B). The mutant is able to cut non-specific ssDNA although with a low activity. The entire Lid region seems to be important for the activity of the protein mutation in this region will induce changes in the activity of the protein. Interestingly, activity of this mutant carrying a substitution of the Lid domain is preserved despite substitution of the E1006. The present inventors have surprisingly found that if the entire Lid domain, including E1006, is substituted activity is preserved provided that the residue corresponding to position 917 of the wild type FnCpf1 is non-substituted, or at the most substituted from an aspartate (D) to a glutamate (E).,” (bottom of page 91 into page 92)
Thus, the art teaches that the entire lid region is important for the function of the protein, where mutations are known to alter the functionality of the protein (Stella, bottom of page 91 to page 92). Thus, there is a higher burden on the Applicant to identify specific mutations within the lid domain which render nickase activity to the recited Cas12a enzymes, as the entirety of the lid domain was known to be critical, as taught by Stella (above). It is suggested that specific mutations in the recited Cas12a enzymes be recited, along with specific sequence information and corresponding locations in the lid domain to address the 112(a) issues of the present claims.
Claims 2, 8-14, and 19-22 depend from claim 1 and do not resolve these 112(a) issues and are therefore also rejected.
Furthermore, regarding claim 2, claim 2 recites Cas12a enzymes with as low as 75% identity to SEQ ID NOs 1-12. As such, claim 2 encompasses mutant variations of Cas12a enzymes such as mutations in AsCas12a (SEQ ID NO: 2). However, it is known in the art that even individual point mutations can have unpredictable effects on Cas12a enzymes. For instance, Kleinstiver (Kleinstiver BP, et al. Nat Biotechnol. 2019 Mar;37(3):276-282) teaches enhanced variants of AsCas12a proteins (Title, Abstract, and throughout). Kleinstiver teaches that they predicted 10 point mutation variants, four of which rendered AsCas12a with higher editing efficiencies of both canonical and non-canonical PAM sites (page 1, right column, second paragraph). Thus, Kleinstiver teaches that individual mutations, let alone variations of 25% of a Cas12a protein, can have profound and unpredictable effects on the functionality of the protein, where furthermore predictions of such alterations are not necessarily reliable and must be tested empirically (i.e., only four of the 10 structure-guided predictive mutations resulted in enhanced editing efficiency at canonical and non-canonical PAM sites for AsCas12a variants, page 1, right column, second paragraph). The Applicant has not sufficiently characterized mutations in other domains of Cas12a proteins, which are reasonably encompassed by claim language such as 75% similarity to SEQ ID NOs 1-12, as presently recited in claim 2.
Furthermore regarding claim 2, the teachings of Kleinstiver, which teaches unpredictable results when obtaining mutations which alter PAM specificity (page 1, right column), speaks to the uncertainty of the claim limitation in claim 2 directed to mutations which alter PAM specificity (final claim limitation of Claim 2, see final four lines of claim 2). The Applicant was not in possession of the genus of mutations which confer altered PAM specificity, because Kleinstiver teaches that empirical mutational analysis is required in order to identify true mutations in a Cas12a enzyme which confer altered PAM specificity, where structure-based guidance is insufficient in itself to determine if a given mutation will confer altered PAM specificity in Cas12a proteins (page 1, right column, second paragraph).
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-2, 8-14, and 19-22 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Stella (WO 2019/233990, A1).
Regarding claim 1, claim 1 is most broadly drawn to a Cas12a comprising a lid domain that is SEQ ID NO: 13 and at least one point mutation. Stella is a patent document which teaches the Cas12a enzyme FnCas12a, as SEQ ID NO: 2, which comprises a lid domain in residues 1006-1018 (page 11, third paragraph). Regarding SEQ ID NO: 2, residues 1006-1022 of SEQ ID NO: 2 (i.e., the lid domain of FnCas12a) are shown below in relation to instant SEQ ID NO: 13:
EDLNFGFKRGRFKVEKQ (Residues 1006-1022, lid domain of SEQ ID NO: 2 of Stella)
LXXGFKXXRXXXXEXXX (SEQ ID NO: 13)
Thus, Stella teaches a Cas12a enzyme which comprises the lid domain of SEQ ID NO: 13, where the Cas12a protein comprises at least a point mutation and/or deletion of the lid domain relative to SEQ ID NO: 13. Furthermore, Stella teaches:
“The FnCpf1 [SEQ ID NO: 2] wild type protein is able to cut both the t-strand and nt-strand (Figures 2-4 and 6). Mutant 1 (SEQ ID NO: 3; K1013-R1014Cpf1) is able to cut only the t-strand (Figures 2, 4 and 6D). This mutant is useful as a nickase,” (page 90, fourth paragraph).
Thus, Stella teaches a mutation in SEQ ID NO: 2 in the lid domain (residues 1013-1014) which render nickase activity to the protein. Thus, Stella anticipates the claim limitations of instant claim 1, as Stella teaches mutations which render nickase activity to a Cas12a, where furthermore relative to SEQ ID NO: 13 the lid domain of the Cas12a comprises at least one point mutation (item “iv” of claim 1) and/or three consecutive point mutations/a deletion/at least one other mutation, as each “X” is mutated to a defined residue in Stella’s SEQ ID NO: 2 lid domain (above).
Regarding claim 2, the limitations of claim 2 are recited in the alternative form. Thus, claim 2 is broadly drawn to the limitations of claim 1 to further include the final limitations recited in claim 2 (“wherein the Cas12a enzyme having nickase activity comprises at least one further mutation…modifies the PAM specificity”). Per the instant specification, the PAM specificity of the Cas12a enzymes recited are TTTV (paragraph 97). Stella recites SEQ ID NO: 2, where SEQ ID NO: 2 is further recited to comprise modified PAM sites relative to the Cas12a enzymes recited in the instant claims (e.g., PAM site recognition of TTN in claim 62 of Stella). Instant SEQ ID NO: 1 and SEQ ID NO: 2 as taught by Stella share ~40% sequence homology (alignment shown below):
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827
1116
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864
1082
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609
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Given that Stella’s SEQ ID NO: 2 is ~40% identical to instant SEQ ID NO: 1 (Applicant’s elected species), it is reasonable to say that Stella’s SEQ ID NO: 2 comprises at least one mutation relative to instant SEQ ID NO: 1. Furthermore, given that Stella teaches that the PAM site is TTN for their Cas12a whereas the PAM sites taught in the present application are TTTV, SEQ ID NO: 2 of Stella “modifies the PAM-specificity” relative to SEQ ID NO: 1, as recited in the final limitation of claim 2.
Regarding claim 8, claim 8 is most broadly drawn to a complex comprising the nickase itself (applicant’ elected embodiment) and a guide RNA. Stella teaches that the nickase can be complexed with a gRNA (e.g., claim 68 of Stella).
Regarding claim 9, claim 9 is most broadly drawn to a Cas12a “fragment” with nicakse activity covalently linked to another polypeptide domain with an enzyme activity. Thus, claim 9 is most broadly drawn to a fragment of the Cas12a nickase covalently linked with another enzymatic domain, which reads on the Cas12a nickase enzyme of claim 1 itself, as Stella teaches that aside from the lid domain, the Cas12a nuclease/nickase is also covalently linked with other enzymatic domains such as the REC and finger domains (page 11, second paragraph).
Regarding claims 10-11, claim 10 recites “a base editor complex…comprising at least one catalytically active portion of at least one engineered Cas12a enzyme having nickas activity of claim 1,” and claim 11 recites “a prime editor or prime editor complex” which comprises a catalytically active portion of the Cas12a enzyme of claim 1. Claims 10 and 11 do not recite further structural requirements or limitations, and are therefore most broadly drawn to the recited components of the complex, namely, a catalytically active portion of the Cas12a enzyme of claim 1, i.e., the Cas12a enzyme of claim 1. These limitations are addressed in the rejection of claim 1, above.
Regarding claim 12, Stella teaches the Cas12a enzyme and guide RNA as discussed in the rejection of claims 1 and 8, above, as presently recited in claim 12. Furthermore, Stella teaches that reactions using the Cas12a and guide RNA can be used in a method to target nucleic acid for cleavage, where such a method reasonably includes “reagents” such as water and the target nucleic acid (claims 113-115 of Stella). Furthermore, recitation of the term “kit” does not add structural significance to the claim. Thus, the reagents of the target nucleic acid, guide RNA, and Cas12a enzyme itself sufficiently read on and anticipate claim 12.
Regarding claims 13-14, Stella teaches the Cas12a enzyme recited in claims 13-14 (see rejection of claim 1, above). Furthermore, Stella teaches that the Cas12a enzyme can be encoded in a nucleic acid molecule that is codon-optimized for expression in E. coli (i.e., a gram-negative prokaryote, page 29, fourth paragraph).
Regarding claim 19, Stella teaches that the nucleic acid can be expressed in a recombinant vector (page 29, fourth paragraph(.
Regarding claim 20, Stella teaches that the gRNA and Cas12a can be expressed from an expression construct/vector (page 29, third paragraph and fourth paragraph). Furthermore, given that Stella teaches that such reagents are used in methods of gene editing, the practitioner can immediately envision that reagents would accompany their use (e.g., water).
Regarding claims 21-22, Stella teaches the Cas12a enzyme recited in claim 21 (see rejection of claim 1). Furthermore, Stella teaches that the Cas12a can be encoded in a construct and expressed in E. coli, a gram-negative prokaryote (page 29, fourth paragraph).
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DOUGLAS CHARLES RYAN whose telephone number is (571)272-8406. The examiner can normally be reached M-F 8AM - 5PM.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram Shukla can be reached at (571)-272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/D.C.R./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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