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 .
Application Status
This action is written in response to applicant’s correspondence received 02/02/2024. Claims 1-4, 7-8, 15-17, 19, 21, 25-26, 30-31, 37, 42 and 60-61 are currently pending.
Specification
The disclosure is objected to because of the following informalities:
Descriptions of the drawings refer to color drawings, although no color drawings are filed with this application. Please see ¶ [00577] and [00580]. The specification should be amended to delete the references to colors in the drawings.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a) – Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(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 1-4, 7-8, 15-17, 19, 21, 25-26, 30-31, 37, 42 and 60-61 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 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 Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Claim 1 is drawn to a variant CRISPR-Cas effector polypeptide with three key characteristics:
1. An amino acid sequence with at least 50% identity to any of SEQ ID NOs: 5-13 and 16-21
2. A deletion or substitution of one or more amino acids in the alpha-7 helix of the RecI domain, compared to either SEQ ID NO: 13 or a corresponding region of another CasPhi polypeptide
3. At least 10% increased cis and/or trans cleave activity compared to a CasPhi polypeptide comprising SEQ ID NO: 13.
The broadest reasonable interpretation of the claimed structure encompasses any amino acid sequence with 50% identity to the recited sequences, with any substitution or deletion of any amino acid or combination of amino acids in the sequence, as long as at least one of those substitutions or deletions occurs at one or more of the residues in the alpha-7 helix of the RecI domain, or a corresponding region of another CasPhi polypeptide. The term “corresponding region” is taken to mean a region which aligns to a known alpha-7 helix in a known RecI region in a reference CasPhi amino acid sequence.
The claim places no limitations on the type or location of any substitution or deletion except that at least one of them must be in the region corresponding to the alpha-7 helix. As a consequence, the deletion(s) or substitution(s) may occur anywhere in the sequence, and the genus of these sequences is correspondingly vast. For example, SEQ ID NO: 20 is 812 amino acids long. A sequence with 50% identity to SEQ ID NO: 20 may have anywhere from 1 to 406 amino acids changed or deleted relative to SEQ ID NO: 20, at any position, in any combination. The claim only requires that at least one This leads to approximately 20406 (1.65E528) possible sequences. The same logic applies to all of the other claimed sequences, which increases the total number of sequences encompassed by the claim commensurately.
In contrast to the breadth of the claimed sequences, the specification discloses two exemplary species of engineered CasPhi variant polypeptide, vCasPhi and nCasPhi, as well as some species of wild type polypeptides which may be used as references.
Regarding the reference sequences, the specification discloses that SEQ ID NO: 13 is a wild-type CasPhi polypeptide (para [0012], FIG. 6). The specification also discloses that SEQ ID NO: 4 is a wild-type CasPhi polypeptide (para [0010], with FIG 4A-4B showing the secondary structure and location of alpha-7). These appear to be two wild-type variants, with the difference that SEQ ID NO: 4 has a deletion in the region corresponding to residues 515-528 of SEQ ID NO: 13, as shown in the below alignment:
GenCore version 6.5.2
Copyright (c) 1993 - 2026 Biocceleration Ltd.
OM protein - protein search, using sw model
Run on: February 11, 2026, 10:56:18 ; Search time 1 Seconds
(without alignments)
0.528 Million cell updates/sec
Title: US-18-273-685-4
Perfect score: 3665
Sequence: 1 AVESEFSKVLKKHFPGERFR..........DVATHNLTQVALTGKTMPKR 698
Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5
Searched: 1 seqs, 757 residues
Total number of hits satisfying chosen parameters: 1
Minimum DB seq length: 0
Maximum DB seq length: inf
Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 1 summaries
Database : US-18-273-685-13.pep:*
SUMMARIES
%
Result Query
No. Score Match Length DB ID Description
----------------------------------------------------------------------------
1 3648 99.5 757 1 US-18-273-685-13 CRISPR-CAS EFFECTO
ALIGNMENTS
RESULT 1
US-18-273-685-13
Query Match 99.5%; Score 3648; DB 1; Length 757;
Best Local Similarity 98.0%;
Matches 698; Conservative 0; Mismatches 0; Indels 14; Gaps 1;
Qy 1 AVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVAYLQGKSEEEPPNFQPPAKC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 5 AVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVAYLQGKSEEEPPNFQPPAKC 64
Qy 61 HVVTKSRDFAEWPIMKASEAIQRYIYALSTTERAACKPGKSSESHAAWFAATGVSNHGYS 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 65 HVVTKSRDFAEWPIMKASEAIQRYIYALSTTERAACKPGKSSESHAAWFAATGVSNHGYS 124
Qy 121 HVQGLNLIFDHTLGRYDGVLKKVQLRNEKARARLESINASRADEGLPEIKAEEEEVATNE 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 125 HVQGLNLIFDHTLGRYDGVLKKVQLRNEKARARLESINASRADEGLPEIKAEEEEVATNE 184
Qy 181 TGHLLQPPGINPSFYVYQTISPQAYRPRDEIVLPPEYAGYVRDPNAPIPLGVVRNRCDIQ 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 185 TGHLLQPPGINPSFYVYQTISPQAYRPRDEIVLPPEYAGYVRDPNAPIPLGVVRNRCDIQ 244
Qy 241 KGCPGYIPEWQREAGTAISPKTGKAVTVPGLSPKKNKRMRRYWRSEKEKAQDALLVTVRI 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 245 KGCPGYIPEWQREAGTAISPKTGKAVTVPGLSPKKNKRMRRYWRSEKEKAQDALLVTVRI 304
Qy 301 GTDWVVIDVRGLLRNARWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFTYTLDACGTY 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 305 GTDWVVIDVRGLLRNARWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFTYTLDACGTY 364
Qy 361 ARKWTLKGKQTKATLDKLTATQTVALVAIDLGQTNPISAGISRVTQENGALQCEPLDRFT 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 365 ARKWTLKGKQTKATLDKLTATQTVALVAIDLGQTNPISAGISRVTQENGALQCEPLDRFT 424
Qy 421 LPDDLLKDISAYRIAWDRNEEELRARSVEALPEAQQAEVRALDGVSKETARTQLCADFGL 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 425 LPDDLLKDISAYRIAWDRNEEELRARSVEALPEAQQAEVRALDGVSKETARTQLCADFGL 484
Qy 481 DPKRLPWDKMSSNTTFISEALLSNSVSRDQVFFT--------------VMRKDRTWARAY 526
|||||||||||||||||||||||||||||||||| ||||||||||||
Db 485 DPKRLPWDKMSSNTTFISEALLSNSVSRDQVFFTPAPKKGAKKKAPVEVMRKDRTWARAY 544
Qy 527 KPRLSVEAQKLKNEALWALKRTSPEYLKLSRRKEELCRRSINYVIEKTRRRTQCQIVIPV 586
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 545 KPRLSVEAQKLKNEALWALKRTSPEYLKLSRRKEELCRRSINYVIEKTRRRTQCQIVIPV 604
Qy 587 IEDLNVRFFHGSGKRLPGWDNFFTAKKENRWFIQGLHKAFSDLRTHRSFYVFEVRPERTS 646
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 605 IEDLNVRFFHGSGKRLPGWDNFFTAKKENRWFIQGLHKAFSDLRTHRSFYVFEVRPERTS 664
Qy 647 ITCPKCGHCEVGNRDGEAFQCLSCGKTCNADLDVATHNLTQVALTGKTMPKR 698
||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 665 ITCPKCGHCEVGNRDGEAFQCLSCGKTCNADLDVATHNLTQVALTGKTMPKR 716
Regarding the exemplary variant species of polypeptide, the specification discloses only two of these which have the function of increased cleavage activity: vCasPhi (para [0013], with the amino acid sequence depicted in SEQ ID NO: 14 and FIG. 7) and nCasPhi (para [0014], SEQ ID NO: 15, FIG. 8). The specification shows a correlation between the structure of these two variants and the function of faster DNA cleavage compared to wild-type CasPhi having the sequence depicted in FIG. 6 (results are shown in e.g., FIG. 3A-3C). Specifically, the structure correlated with that function are either a set of specific alanine substitutions or a deletion of a portion of helix alpha 7:
[00578] Two variant CasPhi polypeptides were engineered, in which helix alpha 7 was modified, either by replacement of amino acids 155-176, or by substitution of E159, S160, S164, D167, and E168. These variant CasPhi polypeptides were designated "vCasPhi" and "nCasPhi." The amino acid sequence of vCasPhi is provided in FIG. 7; as shown in FIG. 7, a GSSG (SEQ ID NO:160) peptide replaces amino acids 155-176 of the wild-type CasPhi-2 amino acid sequence depicted in FIG. 6. The amino acid sequence of nCasPhi is provided in FIG. 8; as shown in FIG. 8, E159, 5160, 5164, D167, and E168 of the amino acid sequence depicted in FIG. 6 are replaced with Ala.
However, the specification does not disclose any other species of CasPhi variants, nor does it disclose any other structure which correlates with the function of at least 10% increased cleavage activity. Here, it is helpful to reiterate that the claim does not particularly limit how a polypeptide sequence may differ from the reference sequence beyond requiring, at a minimum, a deletion or substitution in the region corresponding to the alpha-7 helix of RecI. Therefore, any change is contemplated as long as the overall identity is above 50%, including changes to other regions of the polypeptide outside of the alpha-7 helix. It is also relevant to note that, per FIG. 4B, the alpha-7 helix of RecI spans positions 123-169, i.e., 47 amino acids. If we use SEQ ID NO: 13 as a reference, it is clear that even if the entire alpha-7 helix were to be deleted, the resulting sequence would still have greater than 90% (actually ~94%) identity to SEQ ID NO: 13.
The prior art evidences that the correlation between the structure of CasPhi polypeptides and their cleavage activity was not well-understood in the art, both before and shortly after the filing date of the instant invention. Al Shayeb (Clades of huge phages from across Earth’s ecosystems. Nature 578, 425–431 (2020)) reported the discovery of a new candidate type V effector protein, CasPhi (Cas12j; p. 430 § CRISPR-Cas-mediated interactions), in February 2020. Al Shayeb’s disclosure stated that CasPhi had been detected, but did not disclose any functional characterization. Shortly thereafter, in July 2020, Pausch (CRISPR-Casɸ from huge phages is a hypercompact genome editor.; of record, applicant’s own submission, hereinafter ‘Pausch 2020’) described the Casɸ protein, noting that it had a molecular weight half that of Cas9 and Cas12a (Abstract), and that its, “unusually small size” and, “lack of co-occurring genes raised the question of whether Casɸ functions as a bona fide CRISPR-Cas system” (p. 2). Pausch 2020 further discloses that, “some RuvC domains have been reported to have endoribonucleolytic activity”, and, based on that, they, “tested Casɸ containing a RuvC-inactivating mutation” (p.3), which were D371A and D394A (Fig. 3). They conclude that Casɸ has a single RuvC active site capable of crRNA processing and DNA cutting (p. 4). However, they disclose no further structural information or correlation between the protein’s amino acid sequence and its cleavage function beyond those two sites.
Then, in 2021, Pausch disclosed the cryo-EM structure of Casɸ (DNA interference states of the hypercompact CRISPR-Casɸ effector.; of record, applicant’s submission). The purpose was to “investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA (Abstract). They state that, as of that time, “How Casɸ achieves RNA-guided DNA unwinding and cleavage despite its compact size and evolutionary remoteness from much larger type V CRISPR-Cas proteins is not known.” (p. 2). This evidences that even after the effective filing date of the instant application, the structure of Casɸ was not well-characterized, and the correlation between its structure and its cleavage ability was not understood.
Based on the disclosures of the relatively recent discovery of Casɸ proteins, their significant structural divergence from more well-characterized CRISPR systems, and the stated lack of knowledge regarding the correlation between its structure and its cleavage ability, the prior art evidences that one of ordinary skill would simply not have had the knowledge of Casɸ structure and function to reasonably predict the effect of changes to its amino acid sequence on its cleavage activity.
Given the breadth of the claimed sequences with at least 50% identity to the recited sequences; the disclosure of only two representative species of CasPhi variants with only two very specific changes restricted solely to the alpha-7 helix; the lack of a disclosure of a correlation between any other parts of the CasPhi structure and its cleavage activity; the lack of knowledge in the art about the protein’s structure and how that related to its cleavage activity; and the unpredictability stemming from that lack of knowledge; one of ordinary skill in the art would conclude that based on the breadth of the claims, the limited amount of guidance provided by the specification and the art, the high degree of variation among members of the claimed genus, and further the unpredictability in the art, that one of ordinary skill in the art would conclude that Applicant was not in possession of the invention as broadly claimed.
The dependent claims named in this rejection but not directly addressed are likewise rejected because, while some (e.g., claims 2-4) specify some of the amino acid substitutions, they do not narrow the breadth of the claimed sequences or exclude additional substitutions or deletions beyond those which are recited.
It is noted that when SEQ ID NOs: 14 and 15 (vCasPhi and nCasPhi) are aligned against reference SEQ ID NO: 13, as recited in the claims, they have 96.8% and 99.3% identity to SEQ ID NO: 13, respectively. Therefore, polypeptides with at least 96% amino acid sequence identity to SEQ ID NO: 13, at least 10% increased activity compared to SEQ ID NO: 13, and either of the specific changes to the alpha-7 helix present in those sequences (i.e., the deletion of amino acids 155-176, or substitution of E159, S160, S164, D167, and E168 with alanine) would be supported by the instant specification.
Claim Rejections - 35 USC § 112(a) – Enablement
Claims 60-61 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of modifying a target nucleic acid comprising contacting the target nucleic acid with a vCasPhi (SEQ ID NO: 14) or nCasPhi (SEQ ID NO: 15) variant CRISPR-Cas effector polypeptide, it does not reasonably provide enablement for the method comprising contacting the target nucleic acid with the full scope of any and all polypeptide sequences with at least 50% identity to the sequences recited in claim 1, . The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The test of enablement is whether one skilled in the art could make and use the claimed invention from the disclosures in the specification coupled with information known in the art without undue experimentation (United States v. Telectronics., 8 USPQ2d 1217 (Fed. Cir. 1988)). Whether undue experimentation is needed is not based upon a single factor but rather is a conclusion reached by weighing many factors. These factors were outlined in Ex parte Forman, 230 USPQ 546 (Bd. Pat. App. & Inter. 1986) and again in In re Wands, 8 USPQ2d 1400 (Fed. Cir. 1988), and the most relevant factors are indicated below:
Nature of the Invention
Breadth of the Claims
Guidance of the Specification
State of the Art
Experimentation Required
As discussed in the above rejection of claims 1-3, 7-8, 15-17, 19, 21, 25-26, 30-31, 37, 42 and 60-61 for failure to comply with the written description requirement, the claims encompass a vast genus of CasPhi sequences, but the specification does not disclose a representative number of species and/or a correlation between structure and the function of at least 10% increased cleavage activity sufficient to show possession of the entire genus of polypeptide sequences with at least 50% amino acid identity to the sequences recited in claim 1, from which claims 60-61 depend. Therefore, given the breadth of the claims, state of the art, and the guidance provided by the applicant, as already discussed, the instant specification likewise does not fully enable the claimed method of modifying a target nucleic acid comprising contacting the nucleic acid with the full, generic scope of all the variant CRISPR-Cas effector polypeptides claimed.
Allowable Subject Matter
While the prior art teaches variant CRISPR-Cas effector polypeptides comprising an amino acid sequence having at least 50% amino acid sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 5-13 and 16-21, wherein the variant CRISPR-Cas effector polypeptide comprises a deletion or a substitution of one or more amino acids in the alpha-7 helix of the Rec I domain, compared to the amino acid sequence set forth in SEQ ID NO: 13, or a corresponding region of another CasPhi polypeptide, the prior art does not teach wherein the variant CRISPR-Cas effector polypeptide exhibits at least a 10% increased cis- and/or trans-cleavage activity compared to the cis- and/or trans-cleavage activity of a CasPhi polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13
The closest prior art is WIPO Publication 2020/181101 A1 to Banfield (published 09/10/2020, priority filing date 03/07/2019, applicant’s own submission, shares two inventors with the instant application and has two additional named inventors). Banfield teaches a CRISPR CasPhi polypeptide with 100% identity to instant SEQ ID NO: 8 (SEQ ID NO: 120), as shown below:
RESULT 1
AASEQ2_02242026_093256
Query Match 100.0%; Score 3812; DB 1; Length 717;
Best Local Similarity 100.0%;
Matches 717; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MIKPTVSQFLTPGFKLIRNHSRTAGLKLKNEGEEACKKFVRENEIPKDECPNFQGGPAIA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MIKPTVSQFLTPGFKLIRNHSRTAGLKLKNEGEEACKKFVRENEIPKDECPNFQGGPAIA 60
Qy 61 NIIAKSREFTEWEIYQSSLAIQEVIFTLPKDKLPEPILKEEWRAQWLSEHGLDTVPYKEA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 NIIAKSREFTEWEIYQSSLAIQEVIFTLPKDKLPEPILKEEWRAQWLSEHGLDTVPYKEA 120
Qy 121 AGLNLIIKNAVNTYKGVQVKVDNKNKNNLAKINRKNEIAKLNGEQEISFEEIKAFDDKGY 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 AGLNLIIKNAVNTYKGVQVKVDNKNKNNLAKINRKNEIAKLNGEQEISFEEIKAFDDKGY 180
Qy 181 LLQKPSPNKSIYCYQSVSPKPFITSKYHNVNLPEEYIGYYRKSNEPIVSPYQFDRLRIPI 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 LLQKPSPNKSIYCYQSVSPKPFITSKYHNVNLPEEYIGYYRKSNEPIVSPYQFDRLRIPI 240
Qy 241 GEPGYVPKWQYTFLSKKENKRRKLSKRIKNVSPILGIICIKKDWCVFDMRGLLRTNHWKK 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GEPGYVPKWQYTFLSKKENKRRKLSKRIKNVSPILGIICIKKDWCVFDMRGLLRTNHWKK 300
Qy 301 YHKPTDSINDLFDYFTGDPVIDTKANVVRFRYKMENGIVNYKPVREKKGKELLENICDQN 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 YHKPTDSINDLFDYFTGDPVIDTKANVVRFRYKMENGIVNYKPVREKKGKELLENICDQN 360
Qy 361 GSCKLATVDVGQNNPVAIGLFELKKVNGELTKTLISRHPTPIDFCNKITAYRERYDKLES 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 GSCKLATVDVGQNNPVAIGLFELKKVNGELTKTLISRHPTPIDFCNKITAYRERYDKLES 420
Qy 421 SIKLDAIKQLTSEQKIEVDNYNNNFTPQNTKQIVCSKLNINPNDLPWDKMISGTHFISEK 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 SIKLDAIKQLTSEQKIEVDNYNNNFTPQNTKQIVCSKLNINPNDLPWDKMISGTHFISEK 480
Qy 481 AQVSNKSEIYFTSTDKGKTKDVMKSDYKWFQDYKPKLSKEVRDALSDIEWRLRRESLEFN 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 AQVSNKSEIYFTSTDKGKTKDVMKSDYKWFQDYKPKLSKEVRDALSDIEWRLRRESLEFN 540
Qy 541 KLSKSREQDARQLANWISSMCDVIGIENLVKKNNFFGGSGKREPGWDNFYKPKKENRWWI 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 KLSKSREQDARQLANWISSMCDVIGIENLVKKNNFFGGSGKREPGWDNFYKPKKENRWWI 600
Qy 601 NAIHKALTELSQNKGKRVILLPAMRTSITCPKCKYCDSKNRNGEKFNCLKCGIELNADID 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 NAIHKALTELSQNKGKRVILLPAMRTSITCPKCKYCDSKNRNGEKFNCLKCGIELNADID 660
Qy 661 VATENLATVAITAQSMPKPTCERSGDAKKPVRARKAKAPEFHDKLAPSYTVVLREAV 717
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 VATENLATVAITAQSMPKPTCERSGDAKKPVRARKAKAPEFHDKLAPSYTVVLREAV 717
SEQ ID NO: 120 comprises a deletion or substitution in one or more amino acids corresponding to the alpha-7 helix of the Rec1 domain of SEQ ID NO: 13 (positions 143-189):
RESULT 1
AASEQ2_02242026_093706
Query Match 27.5%; Score 1090.5; DB 1; Length 717;
Best Local Similarity 35.5%;
Matches 266; Conservative 128; Mismatches 295; Indels 61; Gaps 20;
Qy 1 MPKPAVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVAYLQGKS--EEEPPNF 58
| || | |:| || : :: : | | :|||| ::: ::| |||
Db 1 MIKPTV-SQFLT------PGFKLIRNHSRTAGLKLKNEGEEACKKFVRENEIPKDECPNF 53
Qy 59 Q-PPAKCHVVTKSRDFAEWPIMKASEAIQRYIYALSTTERAACKPGKSSESHAAWFAATG 117
| || ::: |||:| || | ::| ||| |: | : :| | | | : |
Db 54 QGGPAIA NIIAKSREFTEWEIYQSSLAIQEVIFTLPKDKLP--EPILKEEWRAQWLSEHG 111
Qy 118 VSNHGYSHVQGLNLIFDHTLGRYDGVLKKVQLRNEKARARLESINASRADEGLPEIKAEE 177
: | ||||| : : | || || :|: |:: | | || ||
Db 112 LDTVPYKEAAGLNLIIKNAVNTYKGVQVKVDNKNKNNLAKINRKNEIAKLNGEQEISFEE 171
Qy 178 EEVATNETGHLLQPPGINPSFYVYQTISPQAY--RPRDEIVLPPEYAGYVRDPNAPIPLG 235
: | :: |:||| | | | | ||::||: : : || || || | | ||
Db 172 IK-AFDDKGYLLQKPSPNKSIYCYQSVSPKPFITSKYHNVNLPEEYIGYYRKSNEPIVSP 230
Qy 236 VVRNRCDIQKGCPGYIPEWQREAGTAISPKTGKAVTVPGLSPKKNKRMRRYWRSEKEKAQ 295
:| | | |||:|:|| || |:||| : |:: |
Db 231 YQFDRLRIPIGEPGYVPKWQ----------------YTFLSKKENKRRK---LSKRIKNV 271
Qy 296 DALLVTVRIGTDWVVIDVRGLLRNARWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFT 355
:| : | || | |:||||| |: |:| | | |||||||| : |:| |
Db 272 SPILGIICIKKDWCVFDMRGLLRTNHWKKYHKPTDSINDLFDYFTGDPVIDTKANVVRFR 331
Qy 356 YTLDACGTYARKWTLKGKQTKATLDKL-TATQTVALVAIDLGQTNPISAGISRVTQENGA 414
| :: | | :: |: | |: : : | :|:|| ||:: |: : : ||
Db 332 YKMEN-GIVNYK-PVREKKGKELLENICDQNGSCKLATVDVGQNNPVAIGLFELKKVNGE 389
Qy 415 LQCEPLDRFTLPDDLLKDISAYRIAWDRNEEELRARSVEALPEAQQAEV-RALDGVSKET 473
| : | | | |:||| :|: | :: ::: | |: || : : :
Db 390 LTKTLISRHPTPIDFCNKITAYRERYDKLESSIKLDAIKQLTSEQKIEVDNYNNNFTPQN 449
Qy 474 ARTQLCADFGLDPKRLPWDKMSSNTTFISE-ALLSNSVSRDQVFFTPAPKKGAKKKAPVE 532
: :|: ::| |||||| | | |||| | :|| : :::|| | | :
Db 450 TKQIVCSKLNINPNDLPWDKMISGTHFISEKAQVSN---KSEIYFTSTDKGKTK-----D 501
Qy 533 VMRKDRTWARAYKPRLSVEAQKLKNEALWALKRTSPEYLKLSRRKEELCRRSINYVIEKT 592
||: | | : |||:|| | : :: | |:| | |: |||: :|: |: |::
Db 502 VMKSDYKWFQDYKPKLSKEVRDALSDIEWRLRRESLEFNKLSKSREQDARQLANWI---- 557
Qy 593 RRRTQCQIVIPVIEDL--NVRFFHGSGKRLPGWDNFFTAKKENRWFIQGLHKAFSDLRTH 650
: | :: ||:| || ||||| ||||||: ||||||:| :||| ::| :
Db 558 --SSMCDVI--GIENLVKKNNFFGGSGKREPGWDNFYKPKKENRWWINAIHKALTELSQN 613
Qy 651 RSFYVFEVRPERTSITCPKCGHCEVGNRDGEAFQCLSCGKTCNADLDVATHNLTQVALTG 710
: | : ||||||||| :|: ||:|| | || || |||:|||| || ||:|
Db 614 KGKRVILLPAMRTSITCPKCKYCDSKNRNGEKFNCLKCGIELNADIDVATENLATVAITA 673
Qy 711 KTMPKREEPRDAQGTAPARKTKKASKSKAP 740
::||| : : |:| :| |:|||
Db 674 QSMPK----PTCERSGDAKKPVRARKAKAP 699
However, Banfield is silent regarding the relative activity of their disclosed variants, including SEQ ID NO: 120. Given how little is known about the correlation between the structure of CasPhi polypeptides and their function, as discussed above in the rejections under 35 U.S.C. § 112(a), the ordinary artisan would not reasonably have been able to predict the cleavage activity of SEQ ID NO: 120 given the information available in Banfield’s specification or what was known in the art.
U.S. Patent No. 11,667,904 B2 to Cheng is the closest prior art which does not share any inventors with the instant application. Sequences disclosed by Cheng have over 50% identity with the claimed sequences, including deletions and/or substitutions in the region corresponding to the alpha-7 helix in SEQ ID NO: 13. For example, SEQ ID NO: 317, an unknown soil metagenome sequence, has 98.6% identity with SEQ ID NO: 6 and has at least one amino acid deletion/substitution in the correct region, as seen below (see also the search results in SCV):
RESULT 1
AASEQ2_02242026_124150
Query Match 30.3%; Score 1203.5; DB 1; Length 761;
Best Local Similarity 36.4%;
Matches 292; Conservative 125; Mismatches 280; Indels 105; Gaps 20;
Qy 3 KPAVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVAYLQGKSEEEPPNFQPPA 62
| | | :|: |||| :| | | || | | |||::|| || : : : :|||
Db 4 KTTPPSPLSLLLRAHFPGLKFESQDYKIAGKKLRDGGPEAVISYLTGKGQAKLKDVKPPA 63
Qy 63 KCHVVTKSRDFAEWPIMKASEAIQRYIYALSTTERAACKPGKS----------------- 105
| |: :|| | || ::: | || |: : |: : | |
Db 64 KAFVIAQSRPFIEWDLVRVSRQIQEKIFGIPATKGRPKQDGLSETAFNEAVASLEVDGKS 123
Qy 106 ---SESHAAWFAATGVSNHGYSHVQGLNLIFDHTLGRYDGVLKKVQLRNEKARARLESIN 162
|: ||:: |: | | | : : :||||||: |||| :::
Db 124 KLNEETRAAFYEVLGLDAPSL-HAQAQNALIKSAISIREGVLKKVENRNEK------NLS 176
Qy 163 ASRADEGLPEIKAEEEEVATNETGHLLQPPGINPSFYVYQTISPQAYRPRDEIVLPPEYA 222
:: : | || | :| |:|: |||:| : || : :: | | | || :
Db 177 KTKRRKEAGEEATFVEEKAHDERGYLIHPPGVNQTIPGYQAVVIKSC-PSDFIGLP---S 232
Qy 223 GYVRDPNAP-----IPLGVVRNRCDIQKGCPGYIPEWQREAGTAISPKTGKAVTVPGLSP 277
| : :| :| :| | || |||:|||| | |:
Db 233 GCLAKESAEALTDYLP----HDRMTIPKGQPGYVPEWQH----------------PLLNR 272
Qy 278 KKNKRMRRYWRS---EKEKA------------------------QDALLVTVRIGTDWVV 310
:||:| || | | | || : |: | :| :||:
Db 273 RKNRR-RRDWYSASLNKPKATCSKRSGTPNRKNSRTDQIQSGRFKGAIPVLMRFQDEWVI 331
Qy 311 IDVRGLLRNARWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFTYTL-DACGTYARKWT 369
||:||||||||:| : : :: || |||||| ||:|: : || | ||
Db 332 IDIRGLLRNARYRKLLKEKSTIPDLLSLFTGDPSIDMRQGVCTFIYKAGQACSAK----M 387
Qy 370 LKGKQTKATLDKLTATQTVALVAIDLGQTNPISAGISRVTQ-ENGALQCEPLDRFTLPDD 428
:| | | :|| : | ||:|||||||||:| :||||| :| | | | | | :|
Db 388 VKTKNAPEILSELTKSGPVVLVSIDLGQTNPIAAKVSRVTQLSDGQLSHETLLRELLSND 447
Qy 429 LL--KDISAYRIAWDRNEEELRARSVEALPEAQQAEVRALDGVSKETARTQLCADFGLDP 486
|:|: ||:| || ::| :|| | ::|: : : ::|| ||:|
Db 448 SSDGKEIARYRVASDRLRDKLANLAVERLSPEHKSEILRAKNDTPALCKARVCAALGLNP 507
Qy 487 KRLPWDKMSSNTTFISEALLSNSVSRDQVFFTPAPKKGAKKKAPVEVMRKDRTWARAYKP 546
: : ||||: | |:: | | | | | | |::|:| :
Db 508 EMIAWDKMTPYTEFLATAYLEKGGDRKVATLKP-------KNRP-EMLRRDIKFKGTEGV 559
Qy 547 RLSV--EAQKLKNEALWALKRTSPEYLKLSRRKEELCRRSINYVIEKTRRRTQCQIVIPV 604
|: | || : || | |:|||||||:|| |:|| :| :| : | : :||::|:
Db 560 RIEVSPEAAEAYREAQWDLQRTSPEYLRLSTWKQELTKRILNQLRHKAAKSSQCEVVVMA 619
Qy 605 IEDLNVRFFHGSGKRLP-GWDNFFTAKKENRWFIQGLHKAFSDLRTHRSFYVFEVRPERT 663
||||:: ||:|| ||| || |:|||||:| ||: ::| |: || | ||
Db 620 FEDLNIKMMHGNGKWADGGWDAFFIKKRENRWFMQAFHKSLTELGAHKGVPTIEVTPHRT 679
Qy 664 SITCPKCGHCEVGNRDGEAFQCLSCGKTCNADLDVATHNLTQVALTGKTMPKREEPR--D 721
|||| |||||: ||||| | | || :|||::|| |: :|||||| ||| | | |
Db 680 SITCTKCGHCDKANRDGERFACQKCGFVAHADLEIATDNIERVALTGKPMPKPESERSGD 739
Qy 722 AQGTAPARKTKKASKSKAPPAE 743
|: : ||| : | ||
Db 740 AKKSVGARKAAFKPEEDAEAAE 761
However, these sequences are largely uncharacterized as far as their functional properties are concerned. Given Cheng’s lack of such a disclosure and the lack of knowledge in the art regarding CasPhi enzymes, as discussed above, the ordinary artisan would not reasonably have predicted that SEQ ID NO: 317 would have had increased cleavage activity relative to SEQ ID NO: 13.
In summary, while the prior art teaches sequences with the required percent identity and one or more deletions/substitutions in the claimed region, the claims also require that the encoded polypeptide have a particular function of increased cleavage activity, and the prior art fails to teach that function or a correlation between structure and function sufficient to guide the ordinary artisan to the claimed sequences.
Conclusion
No claims are allowed at this time.
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/AMANDA M ZAHORIK/Examiner, Art Unit 1636