Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
This application is a 371 of PCT/CN2022/137920.
The amendment filed on June 5, 2026 has been entered.
Election/Restrictions
Applicant’s election without traverse of Group I with a species election of (1) SEQ ID NO:1 as the parent/reference Cas12b nuclease and (2) E475R/K/H as the amino acid modification made in said parent/reference parent/reference Cas12b nuclease in the reply filed on June 5, 2026 is acknowledged.
Claims 13, 19, 29-30, 32-33, 39, 43-44, 56-57, 68, and 70-71 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species (claims 13, 19, 29-30, and 32) and invention (32-33, 39, 43-44, 56-57, 68, and 70-71), there being no allowable generic or linking claim. Election was made without traverse in the reply filed on July 14, 2025.
Applicant’s request for rejoinder has been noted.
Status of Claims
Claims 1, 3, 6, 13, 19, 28-30, 32-33, 39, 43-44, 56-57, 68, and 70-71 are pending.
Claims 13, 19, 29-30, 32-33, 39, 43-44, 56-57, 68, and 70-71 are withdrawn.
Claims 1, 3, 6, and 28 are under examination.
Claim for Foreign Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on December 18, 2024 and June 5, 2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Drawings
The drawings are objected to because FIG 8 is illegible. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
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, 3, 6, and 28 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.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' In the instant case, the claims have been broadly interpreted to encompass any engineered Cas12b nuclease (Cas12b) of any Cas12b or the Cas12b of SEQ ID NO:1, wherein the engineered Cas12b has a substitution of one or more amino acids in the PAM recognition site or the amino acids at the positions recited in claim 6 with a positive amino acid and has any other amino acid modification at any other amino acid positions. Therefore, the claims are drawn to a genus of polypeptide having unknown structure but having Cas12b activity.
MPEP 2163 I. states that 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.
MPEP 2163. II.A.3.(a) sates that “Possession may be shown in many ways. For example, possession may be shown by describing an actual reduction to practice of the claimed invention. Possession may also be shown by a clear depiction of the invention in detailed drawings or in structural chemical formulas which permit a person skilled in the art to clearly recognize that inventor had possession of the claimed invention. An adequate written description of the invention may be shown by any description of sufficient, relevant, identifying characteristics so long as a person skilled in the art would recognize that the inventor had possession of the claimed invention.
According to MPEP 2163.II.A.3.(a).ii), “Satisfactory disclosure of a ‘representative number’ depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus…Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’"
The recitations of “one or more amino acids residue..PAM” and “Cas12b nuclease” fails to provide a sufficient description of the genus of the polypeptides as it merely describes the functional features of the genus without providing any definition of the structural features of the species within the genus. The specification does not specifically define any of the species that fall within the genus. The specification does not define any structural features commonly possessed by members of the genus that distinguish them from others. One skilled in the art therefore cannot, as one can do with a fully described genus, visualize or recognize the identity of the members of the genus.
Alicyclobacillus acidiphilus Cas12b having 100% sequence identity to the Alicyclobacillus acidiphilus Cas12b having the amino acid sequence of SEQ ID NO:1 of the instant application was known in the prior art, see Teng (Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov 4, 63 (2018), Supplementary Information – form PTO-892), NZ_BCQI01000053.1 (GenBank Database. June 29, 2020 – form PTO-892), and see the sequence alignment below. However, engineered Cas12b having any amino acid substitutions of residues in the PAM recognition pocket of any Cas12b or Cas12b of SEQ ID NO:1 and having Cas12b activity were not known in the art.
Fransceus (J Ind Microbiol Biotechnol. 2017 May;44(4-5):687-695. – form PTO-892) reviews protein engineering techniques, such as random mutagenesis and recombination, directed evolution and iterative or combinatory saturation “hotspots”. Fransceus states that “a recurring problem, however, is choosing which amino acid positions should be targeted. Answering this question is not an easy feat and requires substantial insight in the relationship between an enzyme’s sequence or structure and its properties.” Sanavia (Computational and Structural Biotechnology Journal, Volume 18, 2020, Pages 1968-1979. – form PTO-892) discloses challenges in the prediction of protein stability in the occurrence of multiple mutations. “Multiple-point mutations are common variations of the protein sequence that may be needed in protein engineering when a single-point mutation is not enough to yield the desired stability change. Dealing with multiple-site variations adds another level of complexity beyond the prediction of the effect of a single variant on protein stability, since it requires the learning of many types of combinatorial effects”.
The specification is limited to description of specific engineered Cas12b of SEQ ID NO:1 as described in Table 1, such as E475R. While MPEP 2163 acknowledges that in certain situations “one species adequately supports a genus,” it also acknowledges that “[f]or inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus.” In view of the widely variant species encompassed by the genus, the examples of Table 1 described above is not enough and does not constitute a representative number of species to describe the whole genus. Therefore, the specification fails to describe a representative species of the claimed genus.
Further, one of skill in the art could identify mutants of SEQ ID NO:1 or other Cas12b. However, there is no teaching regarding which amino acids can vary from SEQ ID NO:1 or any Cas12b and result in polypeptide having Cas12b activity. An important consideration is that structure is not necessarily a reliable indicator of function. In the instant case, there is no disclosure relating similarity of structure to conservation of function. Conservation of structure is not necessarily a surrogate for conservation of function.
Given this lack of description of the representative species encompassed by the genus of the claims, the specification fails to sufficiently describe the claimed invention in such full, clear, concise, and exact terms that a skilled artisan would recognize that applicants were in possession of the inventions of claims 1, 3, 6, and 28.
Claims 1, 3, 6, and 28 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 specific engineered Cas12b of SEQ ID NO:1 as described in Table 1, such as E475R, does not reasonably provide enablement any polypeptide having unknown structure but having Cas12b activity. 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 use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.
The breadth of the claims.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' In the instant case, the claims have been broadly interpreted to encompass any engineered Cas12b nuclease (Cas12b) of any Cas12b or the Cas12b of SEQ ID NO:1, wherein the engineered Cas12b has a substitution of one or more amino acids in the PAM recognition site or the amino acids at the positions recited in claim 6 with a positive amino acid and has any other amino acid modification at any other amino acid positions. Therefore, the claims are drawn to any polypeptide having unknown structure but having Cas12b activity.
The claims are not commensurate with the enablement provided by the disclosure with regard to the extremely large number of polypeptides having Cas12b activity. In the instant case, the specification is limited to specific engineered Cas12b of SEQ ID NO:1 as described in Table 1, such as E475R.
The quantity of experimentation required to practice the claimed invention based on the teachings of the specification.
While enzyme isolation techniques, recombinant and mutagenesis techniques were known in the art at the time of the invention, e.g. mutagenesis, and it is routine in the art to screen for variants comprising multiple substitutions or multiple modifications as encompassed by the instant claims, the specific amino acid positions within the protein's sequence where amino acid modifications can be made with a reasonable expectation of success in obtaining the desired activity/utility are limited in any protein and the result of such modifications is unpredictable. In addition, one skilled in the art would expect any tolerance to modification for a given protein to diminish with each further and additional modification, e.g. multiple substitutions.
In the absence of: (a) rational and predictable scheme for identifying amino acids in the PAM recognition site of any Cas12b or the Cas12b of SEQ ID NO:1 and substituting said amino acids with a positive amino acid in any Cas12b or the Cas12b of SEQ ID NO:1, and (b) a correlation between structure and the function of having Cas12b activity, the specification provides insufficient guidance as to which of the essentially infinite possible choices is likely to be successful. One of skill in the art would have to test these infinite possible polypeptides to determine amino acids located in the PAM recognition site of any Cas12b or the Cas12b of SEQ ID NO:1 and substituting said amino acids with a positive amino acid in any Cas12b or the Cas12b of SEQ ID NO:1. While enablement is not precluded by the necessity for routine screening, if a large amount of screening is required, as is the case herein, the specification must provide a reasonable amount of guidance which respect to the direction in which the experimentation should proceed so that a reasonable number of species can be selected for testing. In view of the fact that such guidance has not been provided in the instant specification, it would require undue experimentation to enable the full scope of the claims.
The state of prior art, the relative skill of those in the art, and predictability or unpredictability of the art.
Since the amino acid sequence of the mutant determines its structural and functional properties, predictability of which changes can be tolerated in a protein's amino acid sequence and obtain the desired activity requires a knowledge of and guidance with regard to which amino acids in the protein's sequence, if any, are tolerant of modification and which are conserved (i.e. expectedly intolerant to modification), and detailed knowledge of the ways in which the proteins' structure relates to its function. In the instant case, neither the specification or the art provide a correlation between structure and activity such that one of skill in the art can envision the structure of the claimed engineered Cas12b In addition, the art does not provide any teaching or guidance as to (1) which amino acids in any Cas12b are located in the PAM recognition site, (2) which segments of the polypeptide of SEQ ID NO:1 or any Cas12b hat are essential for polypeptides having Cas12b activity, and (3) the general tolerance of any Cas12b or the Cas12b of SEQ ID NO:1 to structural modifications and the extent of such tolerance. The art clearly teaches that changes in a protein's amino acid sequence to obtain the desired activity without any guidance/knowledge as to which amino acids in a protein are required for that activity is highly unpredictable. At the time of the invention there was a high level of unpredictability associated with altering a polypeptide sequence with an expectation that the polypeptide will maintain the desired activity. For example, Studer (Residue mutations and their impact on protein structure and function: detecting beneficial and pathogenic changes. Biochem. J. (2013) 449, 581–594. – form PTO-892) teach that (1) protein engineers are frequently surprised by the range of effects caused by single mutations that they hoped would change only one specific and simple property in enzymes, (2) the often surprising results obtained by experiments where single mutations are made reveal how little is known about the rules of protein stability, and (3) the difficulties in designing de novo stable proteins with specific functions.
Alicyclobacillus acidiphilus Cas12b having 100% sequence identity to the Alicyclobacillus acidiphilus Cas12b having the amino acid sequence of SEQ ID NO:1 of the instant application was known in the prior art, see Teng (Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov 4, 63 (2018). And Supplementary Information – form PTO-892), NZ_BCQI01000053.1 (GenBank Database. June 29, 2020 – form PTO-892), and see the sequence alignment below. However, engineered Cas12b having any amino acid substitutions of residues in the PAM recognition pocket of any Cas12b or Cas12b of SEQ ID NO:1 and having Cas12b activity were not known in the art.
Fransceus (J Ind Microbiol Biotechnol. 2017 May;44(4-5):687-695. – form PTO-892) reviews protein engineering techniques, such as random mutagenesis and recombination, directed evolution and iterative or combinatory saturation “hotspots”. Fransceus states that “a recurring problem, however, is choosing which amino acid positions should be targeted. Answering this question is not an easy feat and requires substantial insight in the relationship between an enzyme’s sequence or structure and its properties.” Sanavia (Computational and Structural Biotechnology Journal, Volume 18, 2020, Pages 1968-1979. – form PTO-892) discloses challenges in the prediction of protein stability in the occurrence of multiple mutations. “Multiple-point mutations are common variations of the protein sequence that may be needed in protein engineering when a single-point mutation is not enough to yield the desired stability change. Dealing with multiple-site variations adds another level of complexity beyond the prediction of the effect of a single variant on protein stability, since it requires the learning of many types of combinatorial effects”.
The amount of direction or guidance presented and the existence of working examples.
The specification is limited to specific engineered Cas12b of SEQ ID NO:1 as described in Table 1, such as E475R. However, the speciation fails to provide any information as to (1) amino acids located in the PAM recognition site of any Cas12b or SEQ ID NO:1 other than those recited in claim 1, (2) specific substrates associated with any Cas12b, and (3) structural elements required in a polypeptide having Cas12b activity.
Thus, in view of the overly broad scope of the claims, the lack of guidance and working examples provided in the specification, the high level of unpredictability of the prior art in regard to structural changes and their effect on function and the lack of knowledge about a correlation between structure and function, an undue experimentation would be necessary one having ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of polypeptides having the desired biological characteristics recited in the claims are unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
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.
Claim(s) 1, 3, 6, and 8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Teng (Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov 4, 63 (2018). And Supplementary Information – form PTO-892), NZ_BCQI01000053.1 (GenBank Database. June 29, 2020 – form PTO-892), Gleditzsch (PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures. RNA Biol. 2019 Apr;16(4):504-517 – form PTO-892), and Yang (PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease. Cell. 2016 Dec 15;167(7):1814-1828 – form PTO-1449)
Regarding claims 1 and 3, Teng discloses Alicyclobacillus acidiphilus Cas12b nuclease (NBRC 100859 AaCas12b) having 100% sequence identity to the AaCas12b nuclease having the amino acid sequence of SEQ ID NO:1 of the instant application (page 2, 2nd full paragraph and “AaCas12b protein sequence” in the “Supplementary Sequences” of the Supplementary Information) and as evidenced by NZ_BCQI01000053.1 (“WP_067623834.1” at page 2 and see the sequence alignment below).
Teng does not disclose substituting amino acids that interact with PAM with a positively charged amino acid residues.
Regarding claim 6, Gleditzsch discloses substituting amino acids of the PAM recognition pocket (amino acids that interact with PAM) of Alicyclobacillus acidoterrestris Cas12a with a positively charged amino acid residues, resulting in expanded range of Cas12a targets (page 511, 2nd paragraph). Gleditzsch discloses that the PAM specificity of the Cas12b is consistent with the TTN PAM of Cas12a (page 511, 3rd paragraph). Gleditzsch discloses that PAM recognition was shown to occur between two domains, termed OBD and Helical-I (page 511, 3rd paragraph). Gleditzsch discloses that residue N144 of Alicyclobacillus acidoterrestris Cas12b is in the PAM recognition pocket (Figure 4 at page 511).
Yang discloses the domains of Alicyclobacillus acidoterrestris Cas12b, which comprises an OBD-I domain (amino acids 1-13), a Helical-1 domain (amino acids 14 to 390), and an OBD-II (amino acids 391-508) (Figure 1A at page 1815).
Regarding claims 6 and 28, Teng also discloses that AaCas12b utilizes TTN PAM for target DNA recognition (page 2, 4th full paragraph). Teng discloses that AaCas12b is comprised of a WED-I domain (also known as OBD-I), a REC-I domain (also known as Helical-I domain), and a WED-II domain (also known as OBD-I) (Fig. 4 at page 7). AaCas12b has 93.5% sequence identity to Alicyclobacillus acidoterrestris Cas12b (see the sequence alignment below). N144 of Alicyclobacillus acidoterrestris Cas12b corresponds to N144 of AaCas12b of SEQ ID NO:1 of the instant application (see the sequence alignment below). E475 of AaCas12b of SEQ ID NO:1 of the instant application correlates to E475 of Alicyclobacillus acidoterrestris Cas12b and resides in the OBD-II domain, part of the PAM recognition pocket (Fig. 4 at page 7 of Teng and Figure 4 at page 511 of Yang).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art at the time the claimed invention was effectively filed to modify Alicyclobacillus acidiphilus Cas12b (AaCas12b) by substituting residues in the PAM recognition pocket, such as N144 and/or E475, with a positively charged residue, Arginine. One having ordinary skill in the art would have been motivated to do so in order to expanded range of AaCas12b targets. One having ordinary skill in the art would have has a reasonable expectation of success since Teng discloses AaCas12b and its WED-I domain (also known as OBD-I), a REC-I domain (also known as Helical-I domain), and a WED-II domain (also known as OBD-I), Gleditzsch discloses substituting amino acids of the PAM recognition pocket of Cas12a with a positively charged amino acid residues, resulting in expanded range of Cas12a targets, Gleditzsch discloses that PAM recognition was shown to occur between two domains (termed OBD and Helical-I), Gleditzsch discloses that residue N144 of Alicyclobacillus acidoterrestris Cas12b is in the PAM recognition pocket, Yang discloses the domains of Alicyclobacillus acidoterrestris Cas12b, which comprises an OBD-I domain (amino acids 1-13), a Helical-1 domain (amino acids 14 to 390), and an OBD-II (amino acids 391-508), Alicyclobacillus acidoterrestris Cas12b high sequence identity to the AaCas12b of SEQ ID NO:1 of the instant application, and N144 and E475 of Alicyclobacillus acidoterrestris Cas12b correlates to N144 and E475 of AaCas12b of SEQ ID NO:1 of the instant application and is in the Helical-1 domain. Using the known technique of replacing amino acids in the PAM recognition pocket individually with an Arginine residue (such as N144R and E475R) in AaCas12b for expanding its range of targets would have been obvious to one of ordinary skill. The rationale supporting that the claims would have been obvious is that a method of enhancing a particular class of devices (replacing amino acids in the PAM recognition pocket individually with an Arginine residue) has been made part of the ordinary capabilities of one skilled in the art based upon the teaching of such improvement in other situations. One of ordinary skill in the art would have been capable of applying this known method of enhancement to a “base” device (AaCas12b) in the prior art and the results would have been predictable to one of ordinary skill in the art.
Therefore, the above references render claims 1, 3, 6, and 8 prima facie obvious.
Claim(s) 1, 3, 6, and 8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Li (CN 113308451 – form PTO-1449) and (Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov 4, 63 (2018). – form PTO-1449), Gleditzsch (PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures. RNA Biol. 2019 Apr;16(4):504-517 – form PTO-892), and Teng (Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov 4, 63 (2018). – form PTO-1449).
Regarding claims 1 and 3, Li discloses Alicyclobacillus acidiphilus Cas12b nuclease (AaCas12b) of SEQ ID NO:7, which has 100% sequence identity to the AaCas12b having the amino acid sequence of SEQ ID NO:1 of the instant application (page see the sequence alignment below). Li discloses that AaCas12b has the following domains: a WEB-I domain (amino acid residue 1-14), REC1 domain (amino acid residue 15-386), WED-II domain (amino acid residue 387-518), RuvC-Istructure domain (amino acid residue 519-628), BH domain (amino acid residue 629-658), REC2domain (amino acid residue 659-784), RuvC-II domain (amino acid residue 785-900), Nuc-I domain(amino acid residue 901-974), RuvC-III domain (amino acid residue 975-993), and Nuc-II domain(amino acid residue 994-1129 994-1129), wherein the amino acid number is based on SEQ ID NO: 7 (page 33, 5th paragraph).
Li does not disclose substituting amino acids that interact with PAM with a positively charged amino acid residues.
Regarding claim 6, Gleditzsch discloses substituting amino acids of the PAM recognition pocket (amino acids that interact with PAM) of Cas12a with a positively charged amino acid residues, resulting in expanded range of Cas12a targets (page 511, 2nd paragraph). Gleditzsch discloses that the PAM specificity of Cas12b is consistent with the TTN PAM of Cas12a (page 511, 3rd paragraph). Gleditzsch discloses that PAM recognition was shown to occur between two domains (termed OBD and Helical-I) ((page 511, 3rd paragraph). Gleditzsch discloses that residue N144 of Alicyclobacillus acidoterrestris Cas12b is in the PAM recognition pocket (Figure 4 at page 511).
Regarding claims 6 and 28, Teng also discloses that AaCas12b utilizes TTN PAM for target DNA recognition (page 2, 4th full paragraph). Teng discloses that AaCas12b is comprised of a WED-I domain (also known as OBD-I), a REC-I domain (also known as Helical-I domain), and a WED-II domain (also known as OBD-I) (Fig. 4 at page 7). AaCas12b has 93.5% sequence identity to Alicyclobacillus acidoterrestris Cas12b (see the sequence alignment below). N144 of Alicyclobacillus acidoterrestris Cas12b corresponds to N144 of AaCas12b of SEQ ID NO:1 of the instant application (see the sequence alignment below). E475 of AaCas12b of SEQ ID NO:1 of the instant application correlates to E475 of Alicyclobacillus acidoterrestris Cas12b and resides in the OBD-II domain, part of the PAM recognition pocket (Fig. 4 at page 7 of Teng).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art at the time the claimed invention was effectively filed to modify Alicyclobacillus acidiphilus Cas12b (AaCas12b) by substituting residues in the PAM recognition pocket with a positively charged residue, Arginine, such as N144R and E475R. One having ordinary skill in the art would have been motivated to do so in order to expanded range of AaCas12b targets. One having ordinary skill in the art would have has a reasonable expectation of success since Li discloses AaCas12b and its WED-I domain (also known as OBD-I), a REC-I domain (also known as Helical-I domain), and a WED-II domain (also known as OBD-I), Gleditzsch discloses substituting amino acids of the PAM recognition pocket of Cas12a with a positively charged amino acid residues, resulting in expanded range of Cas12a targets, and Gleditzsch discloses that PAM recognition was shown to occur between two domains (termed OBD and Helical-I), and Gleditzsch discloses that residue N144 of Alicyclobacillus acidoterrestris Cas12b is in the PAM recognition pocket. Using the known technique of replacing amino acids in the PAM recognition pocket individually with an Arginine residue (such as N144R and E475R) in AaCas12b for expanding its range of targets would have been obvious to one of ordinary skill. The rationale supporting that the claims would have been obvious is that a method of enhancing a particular class of devices (replacing amino acids in the PAM recognition pocket individually with an Arginine residue) has been made part of the ordinary capabilities of one skilled in the art based upon the teaching of such improvement in other situations. One of ordinary skill in the art would have been capable of applying this known method of enhancement to a “base” device (AaCas12b) in the prior art and the results would have been predictable to one of ordinary skill in the art.
Therefore, the above references render claims 1, 3, 6, and 8 prima facie obvious.
Other Relevant Art
CN 113151215 (form PTO-1449) discloses engineered Cas12i, wherein amino acids in the PAM recognition pocket are substituted with positively charged amino acids (abstract and claim 1). However, CN 113151215 discloses that Cas12i, identified as RNA-guided DNA endonuclease system, is different from the CRISPR-Cas system such as Cas12b, because Cas12i does not need tracrRNA sequence (pages 41-42, “6) Reference Cas12i Nuclease”). Zhang (Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease. Nat Struct Mol Biol. 2020 Nov;27(11):1069-1076 – form PTO-1449) discloses that sequence and functional differences between Cas12i and other type V endonucleases indicate that Cas12i may have different molecular mechanisms in target recognition and cleavage (page 1069, 2nd paragraph).
Conclusion
Claims 1, 3, 6, 13, 19, 28-30, 32-33, 39, 43-44, 56-57, 68, and 70-71 are pending.
Claims 13, 19, 29-30, 32-33, 39, 43-44, 56-57, 68, and 70-71 are withdrawn.
Claims 1, 3, 6, and 28 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YONG D PAK whose telephone number is (571)272-0935. The examiner can normally be reached M-Th: 5:30 am - 3:30 pm.
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/YONG D PAK/Primary Examiner, Art Unit 1652
Sequence alignment between the Alicyclobacillus acidiphilus Cas12b of SEQ ID NO:1 of the instant application (“Query”) and the Alicyclobacillus acidiphilus of Teng/NBRC 100859 AaCas12b (“Sbjct”)
PNG
media_image1.png
895
1044
media_image1.png
Greyscale
Sequence alignment between the Alicyclobacillus acidiphilus Cas12b of SEQ ID NO:1 of the instant application (“Qy”) and the Alicyclobacillus acidoterrestris of Teng/NBRC 100859 AaCas12b (“Db”)
CS12B_ALIAG
ID CS12B_ALIAG Reviewed; 1129 AA.
AC T0D7A2; A0A9E6ZFD4;
DT 05-OCT-2016, integrated into UniProtKB/Swiss-Prot.
DT 16-OCT-2013, sequence version 1.
DT 28-JAN-2026, entry version 36.
DE RecName: Full=CRISPR-associated endonuclease Cas12b {ECO:0000303|PubMed:28111461};
DE EC=3.1.-.- {ECO:0000269|PubMed:26593719};
DE AltName: Full=AacC2c1 {ECO:0000303|PubMed:26593719};
DE AltName: Full=CRISPR-associated endonuclease C2c1 {ECO:0000303|PubMed:26593719};
GN Name=cas12b {ECO:0000303|PubMed:28111461};
GN Synonyms=c2c1 {ECO:0000303|PubMed:26593719};
GN ORFNames=K1I37_00465 {ECO:0000312|EMBL:UNO49077.1},
GN N007_06525 {ECO:0000312|EMBL:EPZ47377.1};
OS Alicyclobacillus acidoterrestris (strain ATCC 49025 / DSM 3922 / CIP 106132
OS / NCIMB 13137 / GD3B).
OC Bacteria; Bacillati; Bacillota; Bacilli; Bacillales; Alicyclobacillaceae;
OC Alicyclobacillus.
OX NCBI_TaxID=1356854;
RN [1] {ECO:0000312|EMBL:EPZ47377.1}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B;
RX PubMed=24009113; DOI=10.1128/genomea.00638-13;
RA Shemesh M., Pasvolsky R., Sela N., Green S.J., Zakin V.;
RT "Draft genome sequence of Alicyclobacillus acidoterrestris strain ATCC
RT 49025.";
RL Genome Announc. 5:E00638-E00638(2013).
RN [2] {ECO:0000312|EMBL:UNO49077.1}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B;
RX PubMed=36240455; DOI=10.1093/g3journal/jkac225;
RA Leonardo I.C., Barreto Crespo M.T., Gaspar F.B.;
RT "Unveiling the complete genome sequence of Alicyclobacillus acidoterrestris
RT DSM 3922T, a taint-producing strain.";
RL G3 (Bethesda) 12:0-0(2022).
RN [3]
RP IDENTIFICATION, FUNCTION AS AN ENDONUCLEASE, COFACTOR, BIOPHYSICOCHEMICAL
RP PROPERTIES, AND BIOTECHNOLOGY.
RC STRAIN=ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B;
RX PubMed=26593719; DOI=10.1016/j.molcel.2015.10.008;
RA Shmakov S., Abudayyeh O.O., Makarova K.S., Wolf Y.I., Gootenberg J.S.,
RA Semenova E., Minakhin L., Joung J., Konermann S., Severinov K., Zhang F.,
RA Koonin E.V.;
RT "Discovery and functional characterization of diverse class 2 CRISPR-Cas
RT systems.";
RL Mol. Cell 60:385-397(2015).
RN [4]
RP NOMENCLATURE.
RX PubMed=28111461; DOI=10.1038/nrmicro.2016.184;
RA Shmakov S., Smargon A., Scott D., Cox D., Pyzocha N., Yan W.,
RA Abudayyeh O.O., Gootenberg J.S., Makarova K.S., Wolf Y.I., Severinov K.,
RA Zhang F., Koonin E.V.;
RT "Diversity and evolution of class 2 CRISPR-Cas systems.";
RL Nat. Rev. Microbiol. 15:169-182(2017).
RN [5]
RP REVIEW ON SAFETY OF GENOME EDITING TOOLS.
RX PubMed=36639728; DOI=10.1038/s41467-023-35886-6;
RA Tao J., Bauer D.E., Chiarle R.;
RT "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA
RT editing.";
RL Nat. Commun. 14:212-212(2023).
RN [6] {ECO:0007744|PDB:5U30, ECO:0007744|PDB:5U31, ECO:0007744|PDB:5U33, ECO:0007744|PDB:5U34}
RP X-RAY CRYSTALLOGRAPHY (2.89 ANGSTROMS) IN COMPLEX WITH SGRNA WITH AND
RP WITHOUT TARGET DNA, FUNCTION AS AN ENDONUCLEASE, ACTIVE SITE,
RP BIOPHYSICOCHEMICAL PROPERTIES, DOMAIN, BIOTECHNOLOGY, MUTAGENESIS OF
RP 118-GLN-GLN-119; ARG-122; GLY-143; GLY-478; ARG-507; ASP-570; ARG-574;
RP GLU-848; TYR-853; SER-899; ARG-900; ARG-911 AND ASP-977, DNA-BINDING, AND
RP RNA-BINDING.
RX PubMed=27984729; DOI=10.1016/j.cell.2016.11.053;
RA Yang H., Gao P., Rajashankar K.R., Patel D.J.;
RT "PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas
RT endonuclease.";
RL Cell 167:1814-1828(2016).
RN [7] {ECO:0007744|PDB:5WQE}
RP X-RAY CRYSTALLOGRAPHY (3.13 ANGSTROMS) IN COMPLEX WITH SGRNA, FUNCTION AS
RP AN ENDONUCLEASE, ACTIVE SITE, COFACTOR, SUBUNIT, DOMAIN, BIOTECHNOLOGY,
RP MUTAGENESIS OF TRP-391; GLN-482; ARG-485; ASP-570; ARG-911; ASP-977;
RP ARG-1000 AND ARG-1015, AND RNA-BINDING.
RX PubMed=27989439; DOI=10.1016/j.molcel.2016.11.040;
RA Liu L., Chen P., Wang M., Li X., Wang J., Yin M., Wang Y.;
RT "C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism.";
RL Mol. Cell 65:310-322(2017).
CC -!- FUNCTION: CRISPR (clustered regularly interspaced short palindromic
CC repeat), is an adaptive immune system that provides protection against
CC mobile genetic elements (viruses, transposable elements and conjugative
CC plasmids). CRISPR clusters contain sequences complementary to
CC antecedent mobile elements and target invading nucleic acids. CRISPR
CC clusters are transcribed and processed into CRISPR RNA (crRNA). In type
CC II CRISPR systems correct processing of pre-crRNA requires a trans-
CC encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this
CC protein (By similarity). The tracrRNA serves as a guide for
CC ribonuclease 3-aided processing of pre-crRNA (By similarity). Protein-
CC crRNA-tracrRNA endonucleolytically cleave dsDNA target complementary to
CC the spacer; protein is inactive in the absence of crRNA homologous to
CC the target and tracrRNA (PubMed:26593719). Recognizes a short motif in
CC the CRISPR repeat sequences (the 5' PAM or protospacer adjacent motif,
CC TTN in this organism) to help distinguish self versus nonself, as
CC targets within the bacterial CRISPR locus do not have PAMs
CC (PubMed:26593719). PAM recognition is also required for catalytic
CC activity. Cleavage results in staggered 6-8 base 5'-overhangs 14-17 and
CC 23-24 bases downstream of the PAM (protospacer adjacent motif) on the
CC non-target and target strands respectively (PubMed:27984729,
CC PubMed:27989439). Both target and non-target strand DNA are probably
CC independently cleaved in the same active site (PubMed:27984729).
CC {ECO:0000250|UniProtKB:A0Q5Y3, ECO:0000269|PubMed:26593719,
CC ECO:0000269|PubMed:27984729, ECO:0000269|PubMed:27989439}.
CC -!- COFACTOR:
CC Name=a divalent metal cation; Xref=ChEBI:CHEBI:60240;
CC Evidence={ECO:0000269|PubMed:26593719, ECO:0000269|PubMed:27989439};
CC Note=Optimal in vitro activity occurs in the presence of Mg(2+), but
CC there is weak activity in its absence (PubMed:26593719). A more
CC complete study shows a preference for Mn(2+), although activity is seen
CC with Mg(2+) and Ca(2+) also (PubMed:27989439).
CC {ECO:0000269|PubMed:26593719, ECO:0000269|PubMed:27989439};
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Temperature dependence:
CC Optimum temperature is 50 degrees Celsius cuts between 37 and 60
CC degrees Celsius. {ECO:0000269|PubMed:26593719,
CC ECO:0000269|PubMed:27984729};
CC -!- SUBUNIT: Monomer. {ECO:0000269|PubMed:27989439}.
CC -!- DOMAIN: Structures show 2 discontinuous REC (recognition, residues 15-
CC 386, 658-784) and NUC (nuclease, residues 1-14, 387-658 and 785-1129)
CC lobes composed of several domains each; the boundaries given correspond
CC to Lui et al., but Yang et al., differ only by a few residues in most
CC cases (PubMed:27984729, PubMed:27989439). The crRNA (or single guide,
CC sgRNA) binds in a central channel between the 2 lobes (PubMed:27984729,
CC PubMed:27989439). PAM recognition is sequence specific and occurs
CC mostly via interaction with the REC1 (helical-1) and WED-II (OBD-II)
CC domains (PubMed:27984729). The sgRNA-target DNA heteroduplex binds
CC primarily to the REC lobe in a sequence-independent manner
CC (PubMed:27984729). {ECO:0000305|PubMed:27984729,
CC ECO:0000305|PubMed:27989439}.
CC -!- BIOTECHNOLOGY: The simplicity of the Cas12b-crRNA-tracrRNA endonuclease
CC activity can be used in vitro to target and cleave a DNA sequence of
CC interest, even in the presence of human cell lysate (PubMed:26593719).
CC Fusion of the 5'-tracrRNA:crRNA-3' into a single-guide RNA (sgRNA)
CC leads to a protein-sgRNA system that endonucleolytically cleaves target
CC DNA in vitro (PubMed:26593719, PubMed:27984729, PubMed:27989439).
CC {ECO:0000269|PubMed:26593719, ECO:0000269|PubMed:27984729,
CC ECO:0000269|PubMed:27989439}.
CC -!- MISCELLANEOUS: Part of a type V-B CRISPR-Cas system.
CC {ECO:0000305|PubMed:26593719}.
CC -!- SIMILARITY: Belongs to the CRISPR-associated endonuclease Cas12b
CC family. {ECO:0000305|PubMed:28111461}.
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DR EMBL; AURB01000127; EPZ47377.1; -; Genomic_DNA.
DR EMBL; CP080467; UNO49077.1; -; Genomic_DNA.
DR RefSeq; WP_021296342.1; NZ_AURB01000127.1.
DR PDB; 5U30; X-ray; 2.92 A; A=1-1129.
DR PDB; 5U31; X-ray; 2.89 A; A=1-1129.
DR PDB; 5U33; X-ray; 3.75 A; A=1-1129.
DR PDB; 5U34; X-ray; 3.25 A; A=1-1129.
DR PDB; 5WQE; X-ray; 3.13 A; A=1-1129.
DR PDBsum; 5U30; -.
DR PDBsum; 5U31; -.
DR PDBsum; 5U33; -.
DR PDBsum; 5U34; -.
DR PDBsum; 5WQE; -.
DR AlphaFoldDB; T0D7A2; -.
DR SMR; T0D7A2; -.
DR STRING; 1356854.N007_06525; -.
DR KEGG; aaco:K1I37_00465; -.
DR PATRIC; fig|1356854.4.peg.1703; -.
DR eggNOG; COG0675; Bacteria.
DR OrthoDB; 2369085at2; -.
DR Proteomes; UP000829401; Chromosome.
DR GO; GO:0003677; F:DNA binding; IEA:UniProtKB-KW.
DR GO; GO:0004519; F:endonuclease activity; IEA:UniProtKB-KW.
DR GO; GO:0016787; F:hydrolase activity; IEA:UniProtKB-KW.
DR GO; GO:0003723; F:RNA binding; IEA:UniProtKB-KW.
DR GO; GO:0051607; P:defense response to virus; IEA:UniProtKB-KW.
DR InterPro; IPR054013; C2c1_helical_1st.
DR InterPro; IPR054012; C2c1_helical_2nd.
DR InterPro; IPR054010; C2c1_Nuc-II.
DR InterPro; IPR054011; C2c1_RuvC-like.
DR InterPro; IPR054009; C2c1_WED-II.
DR InterPro; IPR053603; Cas12b_endonuclease.
DR NCBIfam; NF033949; Cas12b; 1.
DR Pfam; PF22172; C2c1_helical; 1.
DR Pfam; PF22202; C2c1_helical_1st; 1.
DR Pfam; PF22077; C2c1_Nuc-II; 1.
DR Pfam; PF22126; C2c1_RuvC-like; 1.
DR Pfam; PF22204; C2c1_WED-II; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antiviral defense; DNA-binding; Endonuclease; Hydrolase;
KW Magnesium; Nuclease; Reference proteome; RNA-binding.
FT CHAIN 1..1129
FT /note="CRISPR-associated endonuclease Cas12b"
FT /id="PRO_0000437504"
FT REGION 1..14
FT /note="WED-I (OBD-I) domain"
FT /evidence="ECO:0000305|PubMed:27984729,
FT ECO:0000305|PubMed:27989439"
FT REGION 4..9
FT /note="Binds sgRNA"
FT /evidence="ECO:0000269|PubMed:27984729,
FT ECO:0000269|PubMed:27989439"
FT REGION 15..386
Query Match 93.5%; Score 5553; Length 1129;
Best Local Similarity 93.7%;
Matches 1059; Conservative 31; Mismatches 38; Indels 2; Gaps 2;
Qy 1 MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQE 60
|||||:|||||||:|||||||||||| |||||||||||||||||||||||||||||||||
Db 1 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQE 60
Qy 61 CYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQI 120
| ||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||
Db 61 CDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQI 120
Qy 121 ARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLR 180
||||||||||||||||||||||||||||||||||||||||||| ||| ||| ||||||||
Db 121 ARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLR 180
Qy 181 ALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGE 240
|||||||||||||||||:||||:||||||||||||||||||||||||||||||||||||:
Db 181 ALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQ 240
Qy 241 AYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSD 300
||||||||:|||||||||||||| ||||||||||||| |||||||||||:|||||||||
Db 241 EYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSD 300
Qy 301 KVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTR 360
|||||| || ||||||||| |||||||||||||||||||||||||:||||||||||||||
Db 301 KVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTR 360
Qy 361 YAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRF 420
|||||||:||||||||||||||||||||||||||||||||||||||||||||| ||||||
Db 361 YAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRF 420
Qy 421 QKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAK 480
||| ||:|||:|||||||||||| |||:||||||:| :||||:||||||| |||||||
Db 421 HKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAK 480
Qy 481 IQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFD 540
|| ||||| |:| ||||||||||:||||||||||||||||||||||||||||||||||||
Db 481 IQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFD 540
Qy 541 KLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPF 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||:|||||
Db 541 KLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPF 600
Qy 601 CFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGS 660
|||:||:||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 FFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGS 660
Qy 661 EDVGRRERSWAKLIEQPMD-ANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVR 719
|||||||||||||||||:| || ||||||||||:||||||||:||| |:|| :|||||||
Db 661 EDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVR 720
Qy 720 RVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKV 779
|||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||
Db 721 RVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKV 780
Qy 780 SGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKY 839
||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||
Db 781 SGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYAL-DERGKGKWVAKY 839
Qy 840 PPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSS 899
||||||||||||||||||||||||||||||||||||||||:|||||||||||||||||||
Db 840 PPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSS 899
Qy 900 RFDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCPLRADDLIPTGEGEF 959
|||||||||||||||||||| :| ||||||||||||| || || |||||||||||||||
Db 900 RFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEI 959
Qy 960 FVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGK 1019
||||||||||||||||||||||||||:||||||||||||||||||||||| ||||| |||
Db 960 FVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGK 1019
Qy 1020 RTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMR 1079
|||||| |||||| |||||||||||||||||||||:||||||||||||||||||||||||
Db 1020 RTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMR 1079
Qy 1080 DPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI 1129
|||||||||:||||||||||||||||||||||||||| ||:|||||||||
Db 1080 DPSGIINRGNWTRQKEFWSMVNQRIEGYLVKQIRSRVPLQDSACENTGDI 1129
Sequence alignment between the Alicyclobacillus acidiphilus Cas12b of SEQ ID NO:1 of the instant application (“Qy”) and the Alicyclobacillus acidiphilus of SEQ ID NO:7 of Li (“Db”)
BJV68456
ID BJV68456 standard; protein; 1129 AA.
XX
AC BJV68456;
XX
DT 28-OCT-2021 (first entry)
XX
DE Alicyclobacillus acidiphilus Cas12b nuclease protein, SEQ 7.
XX
KW C2c1 protein; CRISPR-Cas12b system; Cas12b protein; V-B CRISPR-Cas12b;
KW antibacterial; autoimmune disease; bacterial infection; cancer;
KW cardiovascular disease; cardiovascular-gen.; cell engineering;
KW cytostatic; diagnostic test; dna detection; enzyme engineering;
KW genetic disorder; genetic marker; genetic-disease-gen.; genome editing;
KW immunosuppressive; metabolic disorder; metabolic-gen.;
KW neurodegenerative disease; neuroprotective; ocular disease;
KW ophthalmological; prophylactic to disease; screening; therapeutic;
KW viral infection; virucide.
XX
OS Alicyclobacillus acidiphilus.
XX
CC PN CN113308451-A.
XX
CC PD 27-AUG-2021.
XX
CC PF 07-DEC-2020; 2021CN-10591698.
XX
PR 07-DEC-2020; 2020CN-11414384.
XX
CC PA (CHSC-) CHINESE ACAD SCI ZOOLOGY INST.
CC PA (BEIJ-) BEIJING STEM CELL & REGENERATIVE MED INS.
XX
CC PI Li W, Zhou Q, Chen Y, Hu Y;
XX
DR WPI; 2021-A3529E/084.
XX
CC PT New engineered clustered regularly interspaced short palindromic repeats
CC PT associated protein 12b nuclease used for treating diseases related to
CC PT target nucleic acids in cells of individual, having mutations that
CC PT increases flexibility of flexible region corresponding to reference
CC PT Cas12b nuclease.
XX
CC PS Claim 11; SEQ ID NO 7; 401pp; Chinese.
XX
CC The present invention relates to a novel engineered Cas12b nuclease,
CC useful for treating diseases or disorders related to target nucleic acids
CC in cells of an individual. The Cas12b nuclease comprises one or more
CC mutations that increase the flexibility of the flexible region. The
CC invention further claims: (1) an engineered Cas12b effector protein
CC comprising engineered Cas12b nuclease; (2) an engineered CRISPR-Cas12b
CC system; (3) a method for detecting a target nucleic acid in a sample; (4)
CC a method for modifying a target nucleic acid comprising a target sequence
CC ; and (5) an engineered cell comprising the target nucleic acid modified.
CC The engineered Cas12b nuclease and CRISPR-Cas12b system are useful for
CC treating, preventing, or diagnosing diseases or disorders related to
CC target nucleic acids in cells of the individual, where the diseases or
CC disorders are selected from cancer, cardiovascular disease, genetic
CC disease, autoimmune disease, metabolic disease, neurodegenerative
CC disease, eye disease, bacterial infection, and viral infection by
CC increase Cas12b nuclease enzymatic activity. Note: The present sequence
CC is used as a parent for creating the mutants of (see BJV68683-BJV68685).
XX
SQ Sequence 1129 AA;
Query Match 100.0%; Score 5937; Length 1129;
Best Local Similarity 100.0%;
Matches 1129; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQE 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQE 60
Qy 61 CYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQI 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 CYKTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQI 120
Qy 121 ARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLR 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLR 180
Qy 181 ALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGE 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 ALADFGLKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGE 240
Qy 241 AYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSD 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 AYAKLVEQKSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSD 300
Qy 301 KVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTR 360
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Db 301 KVFEKWEKLDPDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTR 360
Qy 361 YAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRF 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 YAVYNSIVRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRF 420
Qy 421 QKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAK 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 QKLLTVEDGVAKEVDDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAK 480
Qy 481 IQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFD 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 IQYRRDQLNHLHARRGARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFD 540
Qy 541 KLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPF 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 KLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPF 600
Qy 601 CFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGS 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 CFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGS 660
Qy 661 EDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRR 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 EDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRR 720
Qy 721 VWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVS 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 VWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVS 780
Qy 781 GQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYP 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 GQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGKGKWVAKYP 840
Qy 841 PCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSR 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 PCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAAFSSR 900
Qy 901 FDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCPLRADDLIPTGEGEFF 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 FDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCPLRADDLIPTGEGEFF 960
Qy 961 VSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKR 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 VSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKR 1020
Qy 1021 TADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRD 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 TADSYGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRD 1080
Qy 1081 PSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI 1129
|||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 PSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI 1129
Prosecution Insights