DETAILED ACTION
Status of the Application
Claims 41-54 are pending.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendment of claims 41-44 , addition of claims 45-54, and cancellation of claims 24, 27-30, 32-35, 37-40 as submitted in a communication filed on 2/27/2026 is acknowledged.
Applicant elected with traverse Group 53, claim 41, directed in part to a process for inducing targeted meiotic recombination in a eukaryotic cell, wherein said process comprises introducing a fusion protein comprising a Cpf1 nuclease that has SEQ ID NO: 3, and a Spo11 protein that comprises SEQ ID NO: 1 in a communication filed on 8/8/2025.
Claims 45-54 are directed in part to the elected invention. Claims 42-44 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 8/8/2025.
Claims 41, 45-54 is at issue and will be examined to the extent it encompasses the elected invention.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 2/27/2026 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections
Claim 41 remains objected and new claims 45-54 are objected to because they are directed in part to non-elected inventions. Appropriate correction is required.
Claim 49 is objected to due to the recitation of “wherein the amino acid substitution…comprises an alanine substitution”. To enhance clarity and to be consistent with commonly used claim language, the term should be amended to recite “wherein the amino acid substitution…is an alanine substitution”. Appropriate correction is required.
Claim 53 is objected to due to the recitation of “wherein the amino acid substitution…comprises a phenylalanine substitution”. To enhance clarity and to be consistent with commonly used claim language, the term should be amended to recite “wherein the amino acid substitution…is a phenylalanine substitution”. Appropriate correction is required.
Claim Rejections – Improper Markush Grouping
Claim 41 was rejected on the basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). In view of Applicant’s amendment of claim 41, this rejection is hereby withdrawn.
Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA )
Claim 41 remains rejected and new claims 45-54 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
Claim 41 (claims 45-54 dependent thereon) is indefinite in the recitation of “….a sequence that is complementary to a sequence in a targeted chromosome region…” for the following reasons. A nucleotide sequence is a graphical representation of the order in which nucleotides are arranged in a nucleic acid molecule. Therefore, while a chromosome region can comprise a nucleotide sequence, there is no nucleotide sequence in a nucleic acid. If the intended limitation is “..a sequence that is complementary to the sequence of a targeted chromosome region…” the claim should be amended accordingly. Correction is required.
Claim 47 (claim 48-49 dependent thereon) is indefinite in the recitation of “..deficient in nuclease activity as compared to a wild type Cpf1 protein” for the following reasons. The term “a wild type Cfp1 protein” encompasses a genus of proteins. The basis for comparison is variable, thus making a determination as to what is encompassed and excluded from the claim impossible. A protein can be encompassed by the claim if the comparison is made with a wild type Cpf1 protein having SEQ ID NO: X and simultaneously excluded if the comparison is made with a wild type Cpf1 protein having SEQ ID NO: Y. If the intended wild type Cpf1 protein is one that comprises SEQ ID NO: 3, the claim should be amended accordingly. Correction is required.
Claim 51 (claims 52-53 dependent thereon) is indefinite in the recitation of “…deficient in nuclease activity as compared to a wild-type Spo11 protein” for the following reasons. The term “a wild type Spo11 protein” encompasses a genus of proteins. The basis for comparison is variable, thus making a determination as to what is encompassed and excluded from the claim impossible. A protein can be encompassed by the claim if the comparison is made with a wild type Spo11 protein having SEQ ID NO: X and simultaneously excluded if the comparison is made with a wild type Spo11 protein having SEQ ID NO: Y. If the intended wild type Spo11 protein is one that comprises SEQ ID NO: 1, the claim should be amended accordingly. Correction is required.
When amending the claims, applicant is advised to carefully review all examined claims and make the necessary changes to ensure proper antecedent basis and dependency.
Claim Rejections - 35 USC § 112(d) or Fourth Paragraph (pre-AIA )
Claim 41 was rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. In view of Applicant’s amendment, this rejection is hereby withdrawn.
Claim Rejections - 35 USC § 112(a) or First Paragraph (pre-AIA )
Claim 41 remains rejected and new claims 45-54 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention. This rejection as it relates to new claims 45-54 is necessitated by amendment.
This rejection has been discussed at length in the prior Office action. It is maintained and further applied to new claims 45-54 for the reasons for record and those set forth below.
Applicant argues that the recited fusion proteins are adequately described. Applicant states that the Cpf1 protein component of the recited fusion proteins is adequately described. Applicant states that the specification describes the domain structure of Cpf1 proteins as well as several representative Cpf1 proteins from different organisms. Applicant states that the specification also describes how a Cpf1 protein may be modified to be deficient in nuclease activity. With regard to Spo11 proteins, applicant argues that the specification discloses the structure and function of Sp11 proteins and provides several species of Spo11 proteins. Applicant further argues that the specification discloses how a Spo11 protein may be modified to be deficient in nuclease activity. With regard to guide RNAs, Applicant states that guide RNAs for Cpf1 proteins are a 42-44 nucleotide crRNA with a direct repeat sequence of about 19 nucleotides followed by a 23-25 nucleotide protospacer sequence. Applicant states that the guide RNA varies depending on the targeted chromosomal sequence. Applicant states that the specification provides defined structural parameters for guide RNAs and how to introduce them in a eukaryotic cell. Applicant states that the specification adequately describes the architecture of the recited fusion proteins and provides a working example that includes a guide RNA component that associates with the Cpf1 protein.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection or avoid the rejection of new claims 45-54. The Examiner acknowledges the amendments made to the claims, the teachings of the specification as well as the teachings of the prior art. However, the Examiner disagrees with Applicant’s assertion that the entire genus of proteins required in the claimed process are adequately described.
The claims as currently presented require a genus of fusion proteins having (a) any structure, (b) a domain which has at least 80% sequence identity to the polypeptide of SEQ ID NO: 3 and another domain having any structure, and (c) a domain which has at least 80% sequence identity to the polypeptide of SEQ ID NO: 1, and another domain having any structure, wherein said fusion protein comprises (i) a domain which is a Cpf1 protein having nuclease activity or a Cpf1 protein having reduced nuclease activity, and (ii) a domain which is a Spo11 protein with nuclease activity or a Spo11 protein having reduced nuclease activity. See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation.
The Examiner acknowledges that the generic modular arrangement of domains in a Cpf1 protein is known in the art. The Examiner also acknowledges that the prior art and the specification discloses a limited number of Cpf1 proteins and a very limited number of substitutions in a Cpf1 protein that could reduce its endonuclease activity. However, it is noted that neither the specification nor the prior art provide defined structural features required by any protein that is a Cpf1 protein or a structure/function correlation that would allow one of skill in the art to determine which proteins are more likely to be Cpf1 proteins. While the argument can be made that one could use structural homology to determine which proteins are more likely to be Cpf1 proteins, it is noted that there is no disclosure as to the level of structural variability found among all the members of the genus. An alignment of the Cpf1 of SEQ ID NO: 3 and that of SEQ ID NO: 30 shows that they share 37% sequence identity. See alignment below. It is reiterated herein that the specification is silent with regard to the structural features found in the protein of SEQ ID NO: 3 that should be present in any Cpf1 protein, those structural features in the polypeptide of SEQ ID NO: 3 that could be modified without altering function, and the degree of such variability. With regard to the genus of Cpf1 proteins having reduced nuclease activity, it is noted that the claims are not limited to variants of a wild type Cpf1 protein having the few substitutions known in the art to potentially reduce nuclease activity. As such, the genus of Cpf1 proteins having reduced nuclease activity encompass variants of a wild-type Cpf1 protein having any structure and any number of unknown modifications to reduce nuclease activity.
Query = SEQ ID NO:3
Sbjct = SEQ ID NO: 30
NW Score Identities Positives Gaps
2090 523/1430(37%) 770/1430(53%) 187/1430(13%)
Query 1 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF 60
M ++Q+F + Y LSKT+RFEL P +TLE+I A+ + DE A ++K K I+D YH+
Sbjct 1 M-LFQDFTHLYPLSKTVRFELKPIDRTLEHIHAKNFLSQDETMADMHQKVKVILDDYHRD 59
Query 61 FIEEILSSVCISEDLLQNYSDVYFKLKKSD-DDNLQKDFKSAKDTIKKQISEYIKDSEKF 119
FI +++ V +++ L + DVY K +K+ DD LQK K + ++K+I + I + K+
Sbjct 60 FIADMMGEVKLTK--LAEFYDVYLKFRKNPKDDELQKQLKDLQAVLRKEIVKPIGNGGKY 117
Query 120 KNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYF 179
K +++ L AK K K+ G + + L + F+ ++TYF
Sbjct 118 KAGYDR-LFGAKL---------FKDGKELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYF 167
Query 180 KGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLA 239
GFH+NRKN+YS D T+I YR++ +NLP+F++N ++K K + Y+QI +L
Sbjct 168 TGFHDNRKNMYSDEDKHTAIA YRLIHENLPRFIDNLQILTTIKQK--HSALYDQIINELT 225
Query 240 EELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKG 299
D+ + + ++ L Q GIT +NT++GG + K +G
Sbjct 226 AS-GLDVSLAS--------------HLDGYHKLLTQEGITAYNTLLGGISGEAGSPKIQG 270
Query 300 INEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQI 359
INE IN + Q K+ + K+ L KQILSD S SF+ K DDS++ + FY
Sbjct 271 INELINSHHNQHCHKSERIAKLRPLHKQILSDGMSVSFLPSKFADDSEMCQAVNEFYRHY 330
Query 360 A-AFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVL 418
A F V+ LFD + D IY ++ K+L +LS+Q F D++++G +
Sbjct 331 ADVFAKVQS---------LFDGFDDHQKD--GIYVEH-KNLNELSKQAFGDFALLGRVLD 378
Query 419 EYITQQIAPKNLDNPSK-KEQELIAKKT-EKAKYL----SLETIKLALEEFNKHRDIDKQ 472
Y + P+ + +K K AK T EK K++ SL +++ A+E + D D+
Sbjct 379 GYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHYTARHD-DES 437
Query 473 CRFEEILANF----AAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDD-----V 523
+ ++ F A + +I N + + + G++ L + + + +
Sbjct 438 VQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSGKNPEMTQL 497
Query 524 KAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIR 583
+ +K+LLD N+ H K+ K + ++D +FY F Y ELA I LYNK+R
Sbjct 498 RQLKELLDNALNVAHFAKLL-----TTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVR 552
Query 584 NYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDD 643
+Y++QKP+S EK+KLNF N TL NGWD NKE DN ++ KD YYL +++K + K+FD+
Sbjct 553 DYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDN 612
Query 644 KAIKENKGEG-YKKIVYKLLPGANKMLPKVFFSAKSIKF-YNPSEDILRIRNHSTHTKNG 701
N G+ Y+K++YK L K PKVFFS ++I Y+PS++++ I++ +
Sbjct 613 AP---NTGKSIYQKMIYKYLE-VRKQFPKVFFSKEAIA INYHPSKELVEIKDKGRQRSDD 668
Query 702 SPQKGY--------------EKFEFNIEDCRKF--------------------------- 720
K Y +KFE I D + F
Sbjct 669 ERLKLYRFILECLKIHPKYDKKFEGAIGDIQLFKKDKKGREVPISEKDLFDKINGIFSSK 728
Query 721 ------------------------------------------------------IDFYKQ 726
+ +Y +
Sbjct 729 PKLEMEDFFIGEFKRYNPSQDLVDQYNIYKKIDSNDNRKKENFYNNHPKFKKDLVRYYYE 788
Query 727 SISKHPEWKD---FGFRFSDTQRYNSIDEFYREVENQ--GYKLTFENISESYIDSVVNQG 781
S+ KH EW++ F + D Y ++E + E+E + YK++F NI+ YID +V QG
Sbjct 789 SMCKHEEWEESFEFSKKLQDIGCYVDVNELFTEIETRRLNYKISFCNINADYIDELVEQG 848
Query 782 KLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKI 841
+LYLFQIYNKDFS + G+PNLHTLY+KALF E NL D +YKLNGEA++FYRK S+
Sbjct 849 QLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIYKLNGEAQIFYRKASLDMNE 908
Query 842 T--HPAKEAIA NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGAN--KFN 897
T H A E + NKN DNPKK F YD+IKDKR+T+DKF H PIT+NF G +FN
Sbjct 909 TTIHRAGEVLENKNPDNPKKRQ-FVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFN 967
Query 898 DEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNII-----GNDRMKTN 952
++N +++ ++V+++ IDRGERHL Y T+++ KG I++Q + N I +M T
Sbjct 968 KKVNQSIQQY-DEVNVIGIDRGERHLLYLTVINSKGEILEQCSLNDITTASANGTQMTTP 1026
Query 953 YHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGF 1012
YH L E +R +AR W +I IKE+K GYLS VVH+I++L+++YNAIVV EDLNFGF
Sbjct 1027 YHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQISQLMLKYNAIVVLEDLNFGF 1086
Query 1013 KRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTG 1072
KRGRFKVEKQ+YQ E LI+KLN+LV KD D+ G A QLT F K +GKQTG
Sbjct 1087 KRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTG 1146
Query 1073 IIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKN 1132
++YVPA TSKI P TGFV+ L P+YE++++SQ FF KFDKICYN DK YFEF DY
Sbjct 1147 FLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADKDYFEFHIDYAK 1206
Query 1133 FGDKAAKGK--WTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGEC 1190
F DKA + WTI S G + + + + + + EL+ L + I
Sbjct 1207 FTDKAKNSRQIWTICSHGDKRYVYDKTANQNKGAAKGINVNDELKSLFARHHINEKQPNL 1266
Query 1191 IKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM 1250
+ IC +DK+F L +L T+L +R S ++ D+++SPVA+ G FF+S A
Sbjct 1267 VMD-ICQNNDKEFHKSLMYLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQ 1325
Query 1251 PQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 1300
PQ+ADANGAYHI LKGL LL +KN+ + K+ L I N+ + F QNR
Sbjct 1326 PQNADANGAYHIALKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR-- 1373
With regard to a variant having at least 80% sequence identity with the polypeptide of SEQ ID NO: 3, it is noted that such variant allows for any combination of 260 amino acid modifications within the polypeptide of SEQ ID NO: 3 (260 = 0.2x1300; SEQ ID NO: 3 has 1300 amino acids). The total number of variants of a polypeptide having a specific number of amino acid substitutions can be calculated from the formula N!x19A/(N-A)!/A!, where N is the length in amino acids of the reference polypeptide and A is the number of allowed substitutions. Thus, the total number of variants having at least 80% sequence identity with the polypeptide of SEQ ID NO: 3 that result from amino acid substitutions is 1300!x19260/(1300-260)!/260! or 2.73x10613 variants. While the specification discloses a few Cpf1 proteins having nuclease activity and a few Cpf1 proteins having reduced nuclease activity, neither the specification nor the prior art disclose the structural features required in any Cpf1 protein or the structural features within the polypeptide of SEQ ID NO: 3 that are essential to observe the desired nuclease activity. No structure/function correlation has been disclosed which would allow one of skill in the art to determine a priori from an essentially infinite number of variants of the polypeptide of SEQ ID NO: 3 having the required % sequence identity limitation, which ones have the desired nuclease activity.
With regard to the genus of guide RNAs required by the claims, while it is agreed that the specification provides working examples of guide RNAs that can bind to the Cpf1 of SEQ ID NO: 3, and that the generic structure of a Cpf1 guide RNA is known in the art, the specification fails to disclose the specific structural features associated with any guide RNA such that it can bind any Cpf1. It is noted that as known in the art, guide RNAs are specific for a particular CRISPR protein. There is no teaching or suggestion in the specification or the prior art indicating that any Cpf1 protein or variant thereof having reduced nuclease activity will bind to any of the guide RNAs disclosed in the specification. Moreover, there is no indication that the guide RNAs that would bind to the protein of SEQ ID NO: 3 would bind to any structural variant of the polypeptide of SEQ ID NO: 3 having the recited % sequence identity recited. Therefore, in the absence of a structure/function correlation that would allow one of skill in the art to determine which guide RNAs would bind to a particular Cpf1 protein, one cannot reasonably conclude that the entire genus of guide RNAs required by the claims is adequately described.
With regard to Spo11 proteins, the Examiner also acknowledges that the prior art and the specification discloses a limited number of Spo11 proteins and one amino acid substitution that could potentially lead to a reduction in the nuclease activity of a Spo11 protein. However, it is noted that neither the specification nor the prior art provide defined structural features required by any protein that is a Spo11 protein or a structure/function correlation that would allow one of skill in the art to determine which proteins could be Spo11 proteins. There is no disclosure as to the level of structural variability found among all the members of the genus of Spo11 proteins such that one could potentially use structure homology to find those Spo11 proteins required by the claims. An alignment of the spo11 of SEQ ID NO: 1 and that of SEQ ID NO: 20 shows that they share 14% sequence identity. See alignment below. The specification is silent with regard to the structural features found in the protein of SEQ ID NO: 1 that should be present in any Spo11 protein, those structural features in the polypeptide of SEQ ID NO: 1 that could be modified without altering function, and the degree of such variability. While the claims require Spo11 proteins having reduced nuclease activity, it is noted that the claims are not limited to variants of a wild type Spo11 protein having the substitution known in the art to potentially reduce nuclease activity. Thus, the genus of Spo11 proteins having reduced nuclease activity encompass variants of a wild-type Spo11 protein having any structure and any number of unknown modifications to reduce nuclease activity.
Query = SEQ ID NO:1
Sbjct = SEQ ID NO: 20
NW Score Identities Positives Gaps
-139 58/401(14%) 97/401(24%) 195/401(48%)
Query 1 MALEGLRKKYKTRQELVKALTPKRRSIHLNS-NGHSNGTP--CSNADVLAHIKHFLSLAA 57
M ++ TR + I+++ + GT CS + L K +SL
Sbjct 1 MRNASEGQEGFTRSIVEDLARGNAPLIYIDRFRNYCTGTSGNCSCSSGLPTGKEAISLK- 59
Query 58 NSLEQHQQPISIVFQNKKKKGDTSSPDIHTTLDFPLNGPHLCTHQFKLKRCAILLNLLKV 117
+ ++R ILL +L +
Sbjct 60 -------------------------------------------RECHVRRLDILLRVLLI 76
Query 118 VMEKLPLGKNTTVRDIFYSNVELFQRQANVVQWLDVIRFNFKLSPRKSLNIIPAQKGLVY 177
V + L ++ + RDI+Y + +F+ Q+ V + ++ I + S R +LN++
Sbjct 77 VQQLLQENRHGSKRDIYYMHPTVFKEQSVVDRAINDICILLQCS-RHNLNVL-------- 127
Query 178 SPFPIDIYDNILTCENEPKMQKQTIFPGKPCLIPFFQDDAVIKLGTTSMCNIVIVEKEAV 237
+F K D ++ + M N +
Sbjct 128 ----------------------HVLFTVK--------DSKILHIHAFDMGN-----GSSQ 152
Query 238 FTKLVNNYHKLSTNTMLITGKGFPDFLTRLFLKKLEQYCSKLISDCSIFTDADPYGISIA 297
++ HK L G F C IF D SIA
Sbjct 153 VSERPREPHKF-----LALGGEF----------------------CLIFLLHD-LSFSIA 184
Query 298 LNYTHSNERNAYICTMANYKGIRITQVLAQNNEVHNKSIQLLSLNQRDYSLAKNLIASLT 357
E AY+ T VH +SL
Sbjct 185 WGVRCLLEMGAYLYT------------------VH------ISLQ--------------- 205
Query 358 ANSWDIATSPLKNVIIECQREIFFQKKAEMNEIDARIFEYK 398
EC
Sbjct 206 ----------------ECA---------------------S 209
A variant having at least 80% sequence identity with the polypeptide of SEQ ID NO: 1 allows for any combination of 80 amino acid modifications with the polypeptide of SEQ ID NO: 1 (80 = 0.2x398; SEQ ID NO: 1 has 398 amino acids). Using the formula previously provided, the total number of variants having at least 80% sequence identity with the polypeptide of SEQ ID NO: 1 that result from amino acid substitutions is 398!x1980/(398-80)!/80! or 5.39x10187 variants. While the specification discloses a few Spo11 proteins having nuclease activity and a few Spo11 proteins having reduced nuclease activity, neither the specification nor the prior art disclose the structural features required in any Spo11 protein or the structural features within the polypeptide of SEQ ID NO:1 that are essential to observe the desired nuclease activity. No structure/function correlation has been disclosed which would allow one of skill in the art to determine a priori from an essentially infinite number of variants of the polypeptide of SEQ ID NO: 1 having the required % sequence identity limitation, which ones have the desired nuclease activity.
As previously stated, the art teaches several examples of how even highly structurally homologous polypeptides can have different enzymatic activities. See the teachings of Witkowski et al., Tang et al. and Seffernick et al. previously discussed. Since minor structural differences may result in changes affecting function, and no additional information correlating structure with the desired functional characteristics has been provided, one cannot reasonably conclude that the few species disclosed are representative of all the Cpf1 and Spo11 proteins required by the claimed fusion proteins. Therefore, contrary to applicant’s assertions, one of skill in the art cannot reasonably conclude that the entire genus of Cpf1 proteins, Spo11 proteins and guide RNAs are adequately described by the teachings of the specification and/or the prior art.
Claim 41 remains rejected and new claims 45-54 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 process for inducing targeted meiotic recombination by expressing in a eukaryotic cell (i) a fusion protein that comprises the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 2, and (ii) a guide RNA that comprises a region that is complementary to a target DNA and forms a complex with the protein of SEQ ID NO: 3, does not reasonably provide enablement for a process for inducing targeted meiotic recombination by expressing in a eukaryotic cell a fusion protein that has any structure, and a guide RNA having any structure that forms a complex with said fusion protein. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims. This rejection as it relates to new claims 45-54 is necessitated by amendment.
This rejection has been discussed at length in the prior Office action. It is maintained and further applied to new claims 45-54 for the reasons for record and those set forth below.
Applicant argues that the claims have been amended so that the fusion protein requires a Cpf1 protein with nuclease activity or decreased nuclease activity, and a Spo11 protein with nuclease activity or decreased nuclease activity. Applicant states that the specification provides clear and extensive guidance regarding the specific features of Cpf1 proteins, Spo11 proteins and guide RNAs within the scope of the pending claims. According to Applicant, Cpf1 and Spo11 are highly conserved proteins known in the prior art. Therefore, Applicant is of the opinion that one of skill in the art would know which modifications to make to alter nuclease activity. Applicant states that one of skill in the art would readily understand how to select and/or design a guide RNA as required by the claims. Applicant states that the specification provides working examples, as well as specific modifications to reduce nuclease activity in Cpf1 and Spo11 proteins. Applicant states that the specification and the prior art provide a structure/function correlation for both Cpf1 proteins and Spo11 proteins. Applicant states that one of skill in the art would engineer the recited Cpf1 and Spo11 with known and described mutations. Applicant states that the design of guide RNAs for use in the claimed process is routine in the art and based on established principles of molecular biology. Applicant is of the opinion that one of skill in the art can make and use the full scope of the claimed invention without undue experimentation.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection or avoid the rejection of new claims 45-54. The Examiner acknowledges the amendments made to the claims, the teachings of the specification as well as the teachings of the prior art. However, the Examiner disagrees with Applicant’s assertion that the entire scope of the claims is fully enabled.
It is reiterated herein that the claims as currently presented required fusion proteins having (a) any structure, (b) a domain which has at least 80% sequence identity to the polypeptide of SEQ ID NO: 3 and another domain having any structure, or (c) a domain which has at least 80% sequence identity to the polypeptide of SEQ ID NO: 1, and another domain having any structure, wherein said fusion proteins comprise (i) a domain which is a Cpf1 protein having nuclease activity or a Cpf1 protein having reduced endonuclease activity, and (ii) a domain which is a Spo11 protein with nuclease activity or a Spo11 protein having reduced nuclease activity. See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation.
With regard to the argument that the specification provides clear and extensive guidance regarding the specific features of Cpf1 proteins, Spo11 proteins and guide RNAs within the scope of the pending claims, it is noted that while it is agreed that the specification provides a generic description of arrangement of domains in a Cpf1 protein, a few species of Cpf1 and Spo11 proteins, and a very limited number of substitutions in a Cpf1 protein and in Spo11 protein that could reduce their nuclease activity, neither the specification nor the prior art provide defined structural features required by any protein that is a Cpf1 protein or a Spo11 protein, a structure/function correlation that would allow one of skill in the art to determine which proteins are more likely to be Cpf1 proteins or Spo11 proteins, or a structure/function correlation to determine which variants of known Cpf1 and Spo11 proteins are more likely to have reduced nuclease activity. While the claims encompass any Cpf1 or Spo11 protein having reduced nuclease activity, the claims are not limited to variants of a wild type Cpf1 protein or Spo11 protein having the few substitutions known to potentially reduce nuclease activity. As such, the Cpf1 and Spo11 proteins having reduced nuclease activity encompass variants of a wild-type Cpf1 protein and a wild type Spo11 protein having any structure and any number of unknown modifications to reduce nuclease activity.
With regard to the argument that Cpf1 and Spo11 are highly conserved proteins known in the prior art and that one of skill in the art would know which modifications to make to alter nuclease activity, it is reiterated herein that it is clear that Cpf1 proteins and Spo11 proteins are not highly conserved as asserted. As explained above, the Cpf1 proteins of SEQ ID NO: 3 and 30 are only 37% sequence identical while the Spo11 proteins of SEQ ID NO: 1 and 20 are only 14% sequence identical. Therefore, it does not appear that these proteins are highly conserved as asserted. Moreover, while some of the claims require variants of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 1 having at least 80% sequence identity to the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 1, it is noted that the specification and the prior art are silent with regard to the structural features required in these variants such that they can have the desired nuclease activity. While the argument can be made that one could use structural homology to determine which proteins/variants are more likely to be Cpf1 proteins or Spo11 proteins having the desired nuclease activity, it is noted that the art clearly teaches that (a) determining function based solely on structural homology, and (b) modification of a protein’s amino acid sequence to obtain the desired activity without any guidance/knowledge as to which amino acids in a protein are tolerant of modification and which ones are conserved are highly unpredictable. See the teachings of Singh et al. and Sadowski et al. previously discussed. In the absence of some knowledge or guidance as to which variants are more likely to have the desired activity, one of skill in the art would have to test an infinite number of proteins to find those required by the claimed invention. This is not considered routine experimentation.
With regard to the argument that one of skill in the art would readily understand how to select and/or design a guide RNA as required by the claims, it is noted that guide RNAs are specific to the CRISPR protein they form a complex with. While the overall structure of guide RNAs is known, in view of their specificity, one of skill in the art would require some knowledge or guidance as to the specific structural features required for a particular Cpf1 protein. It is reiterated herein that no structure/function correlation has been provided to determine the structure of a guide RNA specific for a particular Cpf1 protein or variant thereof as required by the claims. Therefore, while methods to make polynucleotides are known in the art, one of skill in the art would have to test any number of guide RNAs to find those that would form a complex with a particular Cpf1. It is also noted that while some of the claims require variants of the polypeptide of SEQ ID NO: 3 having at least 80% sequence identity with the polypeptide of SEQ ID NO: 3, it is unclear if all guide RNAs that can form a complex with the polypeptide of SEQ ID NO: 3 would necessarily form a complex with any variant having the recited % sequence identity. In the absence of (i) a rational and predictable scheme for selecting those proteins and guide RNAs most likely to have the desired functional features, and/or (ii) a correlation between structure and the desired activity, one of skill in the art would have to test an essentially infinite number of proteins and guide RNAs to determine which ones have the desired functional characteristics.
Therefore, for the reasons of record and those set forth above, one cannot reasonably conclude that the entire scope of the claimed invention is fully enabled by the teachings of the specification and/or the prior art.
Claim Rejections - 35 USC § 103 (AIA )
Claim 41 remains rejected and new claims 45-54 are rejected under 35 U.S.C. 103 as being unpatentable over Bastianelli et al. (WO 2016/120480 published 8/4/2016; cited in the IDS) in view of Safari et al. (Cell & Bioscience 9:36, pages 1-21, published online 5/9/2019; previously cited) and Miao et al. (Synthetic and systems Biotechnology 4:1-9, published online 11/19/2018). This rejection as it relates to new claims 45-54 is necessitated by amendment.
This rejection has been discussed at length in the prior Office action. It is maintained and further applied to new claims 45-54 for the reasons of record and those set forth below.
Applicant argues that one of skill in the art would have not been motivated to use Cpf1 to induce targeted meiotic recombination and would have not been motivated to substitute a Cas9 protein with a Cpf1 protein. Applicant states that neither Bastianelli et al., Safari et al. nor Miao et al. teach or suggest the possibility of using the CRISPR/Cpf1 system to induce recombination during meiosis of a eukaryotic cell. Applicant states that during meiosis, DNA takes on vary different forms and goes through different processes. Applicant submits that there are significant molecular structural differences in the DNA of a cell during meiosis, citing the teachings of MacGregor et al. (Biochemical Journal 476:2141-2156, 2019; cited in the IDS). Applicant states that cellular physiology is also unique during meiosis, with variations in iron content and intracellular pH, citing Morrill et al. (Biochimica and Biophysica Acta 804:107-117, 1984; cited in the IDS). Applicant is of the opinion that one of skill in the art may have expected that the cellular conditions during meiosis could impact the stability and activity of the Cpf1 complex by leading to changes in the structure of the gRNA and/or Cpf1 protein that are necessary for targeting and DNA binding. Applicant states that specific RNA binding proteins are expressed during meiosis, citing Kasama et al. (Eukaryotic Cell, 2006; cited in the IDS) and that they could interfere with the guide RNA. Applicant states that methylation and compaction can have the ability to negatively impact DNA targeting and binding. Applicant states that Cpf1 produces a double strand break with sticky ends while Cas9 produces clean ends after cleavage. Applicant submits that the guide RNA of a Cpf1 is not the same as that of a Cas9, and that a Cpf1 guide RNA does not require a tracrRNA. Applicant states that these differences would dissuade one of skill in the art from substituting a Cpf1 protein for a Cas9 protein in the context of a meiotic cell. Applicant further argues that the claimed invention is based on unexpected results. Applicant states that it was found that a Cpf1-Sp11 fusion protein system can be used to induce DSBs and promote meiotic recombination. Applicant refers to Figure 1 and 2 in support of the argument that it was unexpected to observe targeted DSBs at the targeted site which were repaired through homologous recombination.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection or avoid the rejection of claims 45-54.
Claims 41, 45-54 as interpreted is directed in part to a process for inducing targeted meiotic recombination which requires expressing (i) a fusion protein that comprise (a) a Cpf1 protein having nuclease activity or reduced nuclease activity, wherein said Cpf1 protein has any structure or has at least 80% sequence identity to the polypeptide of SEQ ID NO: 3, and (b) a Spo11 protein having nuclease activity or reduced nuclease activity, wherein said Spo11 protein has any structure or has at least 80% sequence identity to the polypeptide of SEQ ID NO: 1, and (ii) one or more guide RNAs that form a complex with said Cpf1 protein, wherein the Cpf1 protein having reduced nuclease activity has a substitution corresponding to the substitution D832A in the polypeptide of SEQ ID NO: 4 and the Spo11 protein having reduced nuclease activity has a substitution corresponding to the substitution Y135F in the polypeptide of SEQ ID NO: 1. See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation.
As previously stated Bastianelli et al. teach a method for inducing targeted meiotic recombinations (Abstract; page 1, paragraph [0010]) by introducing into a eukaryotic cell a fusion protein comprising a Cas9 domain and a Spo11 domain and one or more guide RNAs that can form a complex with the Cas9 domain, wherein said one or more guide RNAs have a region which is complementary to a target DNA (page 1, paragraphs [0011]-[0014]), and wherein the Cas9 domain can comprise or consist of a catalytically inactive protein that lacks nuclease activity (page 5, paragraph [0088]; page 3, paragraph [0065]; Figure 14). Bastianelli et al. also teach the introduction of a nucleic acid encoding the fusion protein and the guide RNA in a vector into the eukaryotic cell such that the fusion protein and guide RNA are expressed in said cell (page 1, paragraphs [0018]-[0024]). Bastianelli et al. teach that the Spo11 protein can be from S. cerevisiae (page 5, paragraph [0094]) and that it comprises SEQ ID NO: 9, which is identical to SEQ ID NO: 1 of the instant application. See alignment below. Bastianelli et al. also teach that the Spo11 protein can be a variant of the S. cerevisiae Spo11 protein having the Y135F substitution and that a fusion protein comprising said S. cerevisiae Spo11 variant would have the ability to induce DNA double strand breaks through the Cas9 domain (page 5, paragraph [0094]).
SEQ ID NO:1
BDD06692
ID BDD06692 standard; protein; 398 AA.
XX
AC BDD06692;
XX
DT 22-SEP-2016 (first entry)
XX
DE Saccharomyces cerevisiae SPO11 protein, SEQ ID 9.
XX
KW SPO11 protein; dna detection; dna recombination; meiosis.
XX
OS Saccharomyces cerevisiae.
XX
CC PN WO2016120480-A1.
XX
CC PD 04-AUG-2016.
XX
CC PF 29-JAN-2016; 2016WO-EP052000.
XX
PR 29-JAN-2015; 2015FR-00050707.
PR 22-JUN-2015; 2015FR-00055725.
XX
CC PA (CNRS ) CENT NAT RECH SCI.
CC PA (CURI-) INST CURIE.
CC PA (UPMC ) UNIV CURIE PARIS VI P & M.
CC PA (MEIO-) MEIOGENIX.
XX
CC PI Bastianelli G, Nicolas A, Serero A;
XX
DR WPI; 2016-48037E/56.
XX
CC PT Inducing targeted meiotic recombination in eukaryotic cell, comprises
CC PT introducing fusion protein comprising Cas9 and Spo11 domains and guide
CC PT RNA or nucleic acids encoding them into the cell, and induction of entry
CC PT into prophase I of meiosis.
XX
CC PS Disclosure; SEQ ID NO 9; 66pp; French.
XX
CC The present invention relates to a method for inducing targeted meiotic
CC recombination in an eukaryotic cell. The invention also provides: a
CC fusion protein a Cas9 domain and a Spo11 domain; a nucleic acid encoding
CC the fusion protein; an expression cassette or vector comprising the
CC nucleic acid; a host cell comprising the fusion protein, the nucleic
CC acid, the expression cassette or the vector; a method for generating
CC variants of a eukaryotic organism; a method for identifying or locating
CC the genetic information coding for a characteristic of interest in a
CC eukaryotic cell genome; and a kit comprising the fusion protein, the
CC nucleic acid, the expression cassette, the vector or the host cell. The
CC present sequence represents a Saccharomyces cerevisiae SPO11 protein,
CC which can be used for preparing the kit of the invention.
XX
SQ Sequence 398 AA;
ALIGNMENT:
Query Match 100.0%; Score 2071; Length 398;
Best Local Similarity 100.0%;
Matches 398; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MALEGLRKKYKTRQELVKALTPKRRSIHLNSNGHSNGTPCSNADVLAHIKHFLSLAANSL 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MALEGLRKKYKTRQELVKALTPKRRSIHLNSNGHSNGTPCSNADVLAHIKHFLSLAANSL 60
Qy 61 EQHQQPISIVFQNKKKKGDTSSPDIHTTLDFPLNGPHLCTHQFKLKRCAILLNLLKVVME 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 EQHQQPISIVFQNKKKKGDTSSPDIHTTLDFPLNGPHLCTHQFKLKRCAILLNLLKVVME 120
Qy 121 KLPLGKNTTVRDIFYSNVELFQRQANVVQWLDVIRFNFKLSPRKSLNIIPAQKGLVYSPF 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 KLPLGKNTTVRDIFYSNVELFQRQANVVQWLDVIRFNFKLSPRKSLNIIPAQKGLVYSPF 180
Qy 181 PIDIYDNILTCENEPKMQKQTIFPGKPCLIPFFQDDAVIKLGTTSMCNIVIVEKEAVFTK 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 PIDIYDNILTCENEPKMQKQTIFPGKPCLIPFFQDDAVIKLGTTSMCNIVIVEKEAVFTK 240
Qy 241 LVNNYHKLSTNTMLITGKGFPDFLTRLFLKKLEQYCSKLISDCSIFTDADPYGISIALNY 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 LVNNYHKLSTNTMLITGKGFPDFLTRLFLKKLEQYCSKLISDCSIFTDADPYGISIALNY 300
Qy 301 THSNERNAYICTMANYKGIRITQVLAQNNEVHNKSIQLLSLNQRDYSLAKNLIASLTANS 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 THSNERNAYICTMANYKGIRITQVLAQNNEVHNKSIQLLSLNQRDYSLAKNLIASLTANS 360
Qy 361 WDIATSPLKNVIIECQREIFFQKKAEMNEIDARIFEYK 398
||||||||||||||||||||||||||||||||||||||
Db 361 WDIATSPLKNVIIECQREIFFQKKAEMNEIDARIFEYK 398
As previously indicated, Safari et al. teach that Cpf1 is a CRISPR nuclease that is an alternative to Cas9 because it does not require a tracrRNA and recognizes a 5’-TTV-3’ PAM in a DNA target, thus being an emerging genome editing tool which can be used in various biological approaches (page 2, left column, first full paragraph). Safari et al. teach eight Cpf1 proteins including one from Francisella novicida U112 (page 2 right column, first full paragraph) and the use of DNase deactivated Cpf1 (ddCpf1) as a DNA binding domain to be fused to transcriptional activators (page 9, right column, last two paragraphs-page 11), transcription repressors (page 11, right column-page 12), transcription factors (page 13) and cytidine/adenine deaminases for base editing (page 14, left column). Safari et al. cites reference 12 by Zetcshe et al. (Cell 163(3):759-771, 2015) as disclosing the Cpf1 protein from Francisella novicida U112. As shown in the alignment below, the Cpf1 protein disclosed by Zetsche et al. comprises all of SEQ ID NO: 3. See alignment below.
SEQ ID NO:3
CS12A_FRATN
ID CS12A_FRATN Reviewed; 1300 AA.
AC A0Q7Q2;
DT 09-DEC-2015, integrated into UniProtKB/Swiss-Prot.
DT 09-JAN-2007, sequence version 1.
DT 18-JUN-2025, entry version 79.
DE RecName: Full=CRISPR-associated endonuclease Cas12a {ECO:0000303|PubMed:28111461};
DE EC=3.1.21.1 {ECO:0000269|PubMed:28431230};
DE EC=4.6.1.22 {ECO:0000269|PubMed:28431230};
DE AltName: Full=CRISPR-associated endonuclease Cpf1 {ECO:0000303|PubMed:26422227};
DE AltName: Full=FnCas12a {ECO:0000303|PubMed:28431230};
DE AltName: Full=FnCpf1 {ECO:0000303|PubMed:26422227};
GN Name=cas12a {ECO:0000303|PubMed:28111461};
GN Synonyms=cpf1 {ECO:0000303|PubMed:26422227}; OrderedLocusNames=FTN_1397;
OS Francisella tularensis subsp. novicida (strain U112).
OC Bacteria; Pseudomonadati; Pseudomonadota; Gammaproteobacteria;
OC Thiotrichales; Francisellaceae; Francisella.
OX NCBI_TaxID=401614;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=U112;
RX PubMed=17550600; DOI=10.1186/gb-2007-8-6-r102;
RA Rohmer L., Fong C., Abmayr S., Wasnick M., Larson Freeman T.J., Radey M.,
RA Guina T., Svensson K., Hayden H.S., Jacobs M., Gallagher L.A., Manoil C.,
RA Ernst R.K., Drees B., Buckley D., Haugen E., Bovee D., Zhou Y., Chang J.,
RA Levy R., Lim R., Gillett W., Guenthener D., Kang A., Shaffer S.A.,
RA Taylor G., Chen J., Gallis B., D'Argenio D.A., Forsman M., Olson M.V.,
RA Goodlett D.R., Kaul R., Miller S.I., Brittnacher M.J.;
RT "Comparison of Francisella tularensis genomes reveals evolutionary events
RT associated with the emergence of human pathogenic strains.";
RL Genome Biol. 8:R102.1-R102.16(2007).
RN [2]
RP FUNCTION IN PLASMID RESISTANCE, FUNCTION IN CRRNA FORMATION, FUNCTION AS AN
RP ENDONUCLEASE, POSSIBLE ACTIVE SITE, COFACTOR, POSSIBLE SUBUNIT, AND
RP MUTAGENESIS OF ASP-917; GLU-1006 AND ASP-1255.
RC STRAIN=U112;
RX PubMed=26422227; DOI=10.1016/j.cell.2015.09.038;
RA Zetsche B., Gootenberg J.S., Abudayyeh O.O., Slaymaker I.M., Makarova K.S.,
RA Essletzbichler P., Volz S.E., Joung J., van der Oost J., Regev A.,
RA Koonin E.V., Zhang F.;
RT "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.";
RL Cell 163:759-771(2015).
RN [3]
RP DISCUSSION OF SEQUENCE.
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 FUNCTION IN CRRNA PROCESSING, FUNCTION AS AN ENDORIBONUCLEASE, FUNCTION AS
RP AN ENDONUCLEASE, COFACTOR, SUBUNIT, MUTAGENESIS OF HIS-843; LYS-852;
RP LYS-869; PHE-873; ASP-917; GLU-920; HIS-922; TYR-925; GLU-1006; TYR-1024;
RP GLU-1028; ASP-1227 AND ASP-1255, DNA-BINDING, AND RNA-BINDING.
RC STRAIN=U112;
RX PubMed=27096362; DOI=10.1038/nature17945;
RA Fonfara I., Richter H., Bratovic M., Le Rhun A., Charpentier E.;
RT "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor
RT CRISPR RNA.";
RL Nature 532:517-521(2016).
RN [5]
RP BIOTECHNOLOGY.
RX PubMed=27905529; DOI=10.1038/srep38169;
RA Endo A., Masafumi M., Kaya H., Toki S.;
RT "Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from
RT Francisella novicida.";
RL Sci. Rep. 6:38169-38169(2016).
RN [6]
RP BIOTECHNOLOGY.
RX PubMed=27272384; DOI=10.1038/nbt.3609;
RA Kim D., Kim J., Hur J.K., Been K.W., Yoon S.H., Kim J.S.;
RT "Genome-wide analysis reveals specificities of Cpf1 endonucleases in human
RT cells.";
RL Nat. Biotechnol. 34:863-868(2016).
RN [7]
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 [8]
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 [9] {ECO:0007744|PDB:5NFV, ECO:0007744|PDB:5NG6}
RP X-RAY CRYSTALLOGRAPHY (2.50 ANGSTROMS) OF 2-1300 IN COMPLEX WITH GUIDE RNA
RP WITH AND WITHOUT TARGET DNA, FUNCTION IN CRRNA PROCESSING, FUNCTION AS AN
RP ENDONUCLEASE, ACTIVE SITE, CATALYTIC ACTIVITY, REACTION MECHANISM,
RP COFACTOR, SUBUNIT, DOMAIN, MUTAGENESIS OF ARG-692; 689-THR--SER-702;
RP GLN-704; ASP-917; GLU-1006; ARG-1218 AND ASP-1255, DNA-BINDING, AND
RP RNA-BINDING.
RC STRAIN=U112;
RX PubMed=28431230; DOI=10.1016/j.molcel.2017.03.016;
RA Swarts D.C., van der Oost J., Jinek M.;
RT "Structural basis for guide RNA processing and seed-dependent DNA targeting
RT by CRISPR-Cas12a.";
RL Mol. Cell 66:221-233(2017).
RN [10] {ECO:0007744|PDB:5MGA}
RP X-RAY CRYSTALLOGRAPHY (3.00 ANGSTROMS) IN COMPLEX WITH GUIDE RNA AND
RP PRODUCT DNA, FUNCTION AS AN ENDONUCLEASE, COFACTOR, DOMAIN, MUTAGENESIS OF
RP GLY-608; PRO-663; ASN-666; LYS-667; LYS-671; LYS-677; ARG-692; HIS-694;
RP GLU-1006; 1065-LYS-LYS-1066 AND ARG-1218, DNA-BINDING, AND RNA-BINDING.
RC STRAIN=U112;
RX PubMed=28562584; DOI=10.1038/nature22398;
RA Stella S., Alcon P., Montoya G.;
RT "Structure of the Cpf1 endonuclease R-loop complex after target DNA
RT cleavage.";
RL Nature 546:559-563(2017).
RN [11]
RP ERRATUM OF PUBMED:28562584.
RX PubMed=28678773; DOI=10.1038/nature23300;
RA Stella S., Alcon P., Montoya G.;
RT "Structure of the Cpf1 endonuclease R-loop complex after target DNA
RT cleavage.";
RL Nature 547:476-476(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). Has
CC endonuclease activity on pre-crRNA and dsDNA, using different active
CC sites. A single-RNA guided endonuclease that is also capable of guiding
CC crRNA processing; correct processing of pre-crRNA requires only this
CC protein and the CRISPR locus (PubMed:26422227, PubMed:27096362). pre-
CC crRNA processing proceeds by an intramolecular nucleophilic attack on
CC the scissile phosphate by the 2'-OH of the upstream ribonucleotide, the
CC divalent cation (which is bound by the crRNA) is probably required for
CC ordering the crRNA pseudoknot and/or increasing RNA binding
CC (PubMed:28431230). RNA mutagenesis studies show pre-crRNA cleavage is
CC highly sequence- and structure-specific (PubMed:27096362). Forms a
CC complex with crRNA and complementary dsDNA, where the crRNA displaces
CC the non-target DNA strand and directs endonucleolytic cleavage of both
CC strands of the DNA (PubMed:26422227, PubMed:27096362, PubMed:28431230).
CC Cleavage results in staggered 5-base 5' overhangs 14-18 and 21-23 bases
CC downstream of the PAM (protospacer adjacent motif) on the non-target
CC and target strands respectively (PubMed:26422227, PubMed:28431230,
CC PubMed:28562584). Both target and non-target strand DNA are probably
CC independently cleaved in the same active site (PubMed:28431230,
CC PubMed:28562584). When this protein is expressed in E.coli it prevents
CC plasmids homologous to the first CRISPR spacer from transforming,
CC formally showing it is responsible for plasmid immunity
CC (PubMed:26422227). {ECO:0000269|PubMed:26422227,
CC ECO:0000269|PubMed:27096362, ECO:0000269|PubMed:28431230,
CC ECO:0000269|PubMed:28562584}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=Endonucleolytic cleavage to 5'-phosphodinucleotide and 5'-
CC phosphooligonucleotide end-products.; EC=3.1.21.1;
CC Evidence={ECO:0000269|PubMed:28431230};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=RNA = a 5'-hydroxy-ribonucleotide + n nucleoside-2',3'-
CC cyclophosphates.; EC=4.6.1.22;
CC Evidence={ECO:0000269|PubMed:28431230};
CC -!- COFACTOR:
CC Name=Ca(2+); Xref=ChEBI:CHEBI:29108;
CC Evidence={ECO:0000269|PubMed:26593719};
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420;
CC Evidence={ECO:0000269|PubMed:26422227, ECO:0000269|PubMed:26593719,
CC ECO:0000269|PubMed:27096362, ECO:0000269|PubMed:28562584};
CC Note=Cleavage of dsDNA requires Mg(2+) (PubMed:26422227,
CC PubMed:28562584). Another report shows DNA cleavage occurs equally well
CC in the presence of Ca(2+) or Mg(2+) (PubMed:27096362). Processing of
CC pre-crRNA requires a divalent cation, preferably Mg(2+) which is bound
CC by the crRNA (PubMed:26593719, PubMed:28431230).
CC {ECO:0000269|PubMed:26422227, ECO:0000269|PubMed:26593719,
CC ECO:0000269|PubMed:27096362, ECO:0000269|PubMed:28431230,
CC ECO:0000269|PubMed:28562584};
CC -!- SUBUNIT: Might be a homodimer (PubMed:26422227). Might be a monomer
CC (PubMed:27096362, PubMed:28431230). {ECO:0000269|PubMed:26422227,
CC ECO:0000269|PubMed:27096362, ECO:0000269|PubMed:28431230}.
CC -!- DOMAIN: Has bilobed structure, with the REC lobe (residues 25-591)
CC connected to the NUC lobe (662-1300) by a discontinuous wedge domain
CC (PubMed:28431230, PubMed:28562584). The REC lobe binds the (pre-)crRNA
CC and the crRNA-target DNA heteroduplex (PubMed:28431230,
CC PubMed:28562584). The heteroduplex as well as part of the DNA
CC downstream of the heteroduplex is protected in the central cavity
CC formed by the NUC and REC lobes, which also positions target and non-
CC target DNA for cleavage after domain rearrangement (PubMed:28431230,
CC PubMed:28562584). The LKL region (residues 662 to 679) inserts into
CC target dsDNA initiating its disruption to allow crRNA hybridization, is
CC also involved in determining the non-target strand cleavage site
CC (PubMed:28562584). A 'septum' formed by residues 197-204 and 1061-1070
CC separates the 2 DNA strands, preventing their reannealing, this region
CC also influences the non-target cleavage site (PubMed:28562584).
CC {ECO:0000269|PubMed:28431230, ECO:0000269|PubMed:28562584}.
CC -!- BIOTECHNOLOGY: This class of CRISPR enzymes recognize a 5' T-rich
CC protospacer adjacent motif (PAM, TTN for this specific enzyme), unlike
CC Cas9 enzymes which recognize 3' G-rich PAMs, thus this enzyme increases
CC the possibilites for genome editing (PubMed:26422227). The simplicity
CC of the Cas12a-crRNA directed DNA endonuclease activity has been used to
CC target and modify DNA sequences in rice and tobacco (PubMed:27905529).
CC {ECO:0000269|PubMed:27272384, ECO:0000269|PubMed:27905529,
CC ECO:0000305|PubMed:26422227}.
CC -!- MISCELLANEOUS: Part of a type V-A CRISPR-Cas system.
CC {ECO:0000305|PubMed:26593719, ECO:0000305|PubMed:28111461}.
CC -!- SIMILARITY: Belongs to the CRISPR-associated endonuclease Cas12a
CC family. {ECO:0000305|PubMed:28111461}.
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DR EMBL; CP000439; ABK90267.1; -; Genomic_DNA.
DR RefSeq; WP_003040289.1; NC_008601.1.
DR PDB; 5MGA; X-ray; 3.00 A; A=1-1300.
DR PDB; 5NFV; X-ray; 2.50 A; A=2-1300.
DR PDB; 5NG6; X-ray; 3.34 A; A/C/E/G=2-1300.
DR PDB; 6GTC; EM; 3.91 A; A=1-1300.
DR PDB; 6GTD; EM; 4.24 A; A=1-1300.
DR PDB; 6GTE; EM; 4.07 A; A=1-1300.
DR PDB; 6GTF; EM; 3.63 A; A=1-1300.
DR PDB; 6GTG; EM; 3.27 A; A=1-1300.
DR PDB; 6I1K; X-ray; 2.65 A; A=2-1300.
DR PDB; 6I1L; X-ray; 2.98 A; A/D=2-1300.
DR PDB; 8Y0B; X-ray; 2.30 A; A=1-1300.
DR PDB; 8Y0C; X-ray; 3.45 A; A=1-1300.
DR PDBsum; 5MGA; -.
DR PDBsum; 5NFV; -.
DR PDBsum; 5NG6; -.
DR PDBsum; 6GTC; -.
DR PDBsum; 6GTD; -.
DR PDBsum; 6GTE; -.
DR PDBsum; 6GTF; -.
DR PDBsum; 6GTG; -.
DR PDBsum; 6I1K; -.
DR PDBsum; 6I1L; -.
DR PDBsum; 8Y0B; -.
DR PDBsum; 8Y0C; -.
DR AlphaFoldDB; A0Q7Q2; -.
DR EMDB; EMD-0061; -.
DR EMDB; EMD-0062; -.
DR EMDB; EMD-0063; -.
DR EMDB; EMD-0064; -.
DR EMDB; EMD-0065; -.
DR SMR; A0Q7Q2; -.
DR KEGG; ftn:FTN_1397; -.
DR KEGG; ftx:AW25_605; -.
DR BioCyc; FTUL401614:G1G75-1444-MONOMER; -.
DR Proteomes; UP000000762; Chromosome.
DR GO; GO:0004530; F:deoxyribonuclease I activity; IEA:UniProtKB-EC.
DR GO; GO:0003677; F:DNA binding; IEA:UniProtKB-KW.
DR GO; GO:0016829; F:lyase 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; IPR027620; Cas12a.
DR InterPro; IPR040882; Cas12a_NUC.
DR InterPro; IPR053993; Cas12a_PI.
DR InterPro; IPR040787; Cas12a_REC1.
DR InterPro; IPR054116; Cas12a_REC2.
DR InterPro; IPR040852; RuvC_1.
DR NCBIfam; TIGR04330; cas_Cpf1; 1.
DR Pfam; PF21918; cas_Cpf1_2nd; 1.
DR Pfam; PF22222; Cpf1_PI-like; 1.
DR Pfam; PF18510; NUC; 1.
DR Pfam; PF18501; REC1; 1.
DR Pfam; PF18516; RuvC_1; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antiviral defense; Calcium; DNA-binding; Endonuclease;
KW Hydrolase; Lyase; Magnesium; Nuclease; RNA-binding.
FT CHAIN 1..1300
FT /note="CRISPR-associated endonuclease Cas12a"
FT /id="PRO_0000434902"
FT REGION 1..24
FT /note="Wedge region 1"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 25..339
FT /note="Recognition domain 1"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 47..51
FT /note="Binds crRNA alone and in crRNA-target DNA
FT heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT REGION 182..186
FT /note="Binds crRNA alone and in crRNA-target DNA
FT heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT REGION 301..305
FT /note="Binds DNA in crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230"
FT REGION 326..329
FT /note="Binds crRNA in crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT REGION 340..591
FT /note="Recognition domain 2"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 538..541
FT /note="Binds crRNA in crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230"
FT REGION 591..595
FT /note="Binds crRNA"
FT /evidence="ECO:0000269|PubMed:28431230"
FT REGION 592..662
FT /note="Wedge region 2"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 662..679
FT /note="LKL, important for PAM recognition and DNA
FT unwinding"
FT /evidence="ECO:0000305|PubMed:28562584"
FT REGION 663..762
FT /note="PAM-interacting domain (PI)"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 671..677
FT /note="Binds DNA protospacer adjacent motif (PAM) on target
FT DNA"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT REGION 692..704
FT /note="Binds single-strand non-target DNA"
FT /evidence="ECO:0000269|PubMed:28431230"
FT REGION 763..892
FT /note="Wedge region 3"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 791..794
FT /note="Binds crRNA"
FT /evidence="ECO:0000269|PubMed:28431230"
FT REGION 803..804
FT /note="Binds crRNA"
FT /evidence="ECO:0000269|PubMed:28431230"
FT REGION 851..853
FT /note="Binds crRNA"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT REGION 865..873
FT /note="Binds crRNA"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT REGION 893..953
FT /note="RuvC-I"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 954..971
FT /note="Bridge helix"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 972..1078
FT /note="RuvC-II"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 1079..1254
FT /note="Nuclease domain"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT REGION 1255..1300
FT /note="RuvC-III"
FT /evidence="ECO:0000303|PubMed:28431230,
FT ECO:0000303|PubMed:28562584"
FT ACT_SITE 843
FT /note="For pre-crRNA processing"
FT /evidence="ECO:0000305|PubMed:28431230"
FT ACT_SITE 852
FT /note="For pre-crRNA processing"
FT /evidence="ECO:0000305|PubMed:28431230"
FT ACT_SITE 869
FT /note="For pre-crRNA processing"
FT /evidence="ECO:0000305|PubMed:28431230"
FT ACT_SITE 917
FT /note="For DNase activity of RuvC domain"
FT /evidence="ECO:0000305|PubMed:26422227"
FT ACT_SITE 1006
FT /note="For DNase activity of RuvC domain"
FT /evidence="ECO:0000305|PubMed:26422227"
FT ACT_SITE 1255
FT /note="For DNase activity of RuvC domain"
FT /evidence="ECO:0000305|PubMed:26422227"
FT SITE 16
FT /note="Binds crRNA alone and in crRNA-target DNA
FT heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT SITE 131
FT /note="Binds target strand DNA"
FT /evidence="ECO:0000269|PubMed:28431230"
FT SITE 295
FT /note="Binds crRNA in crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230"
FT SITE 320
FT /note="Binds DNA in crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230"
FT SITE 334
FT /note="Binds DNA in crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230"
FT SITE 410
FT /note="Caps the crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000305|PubMed:28431230"
FT SITE 589
FT /note="Binds DNA in crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230"
FT SITE 613
FT /note="Binds DNA protospacer adjacent motif (PAM)"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT SITE 667
FT /note="Binds Target strand DNA"
FT /evidence="ECO:0000269|PubMed:28431230"
FT SITE 671
FT /note="Binds PAM"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT SITE 677
FT /note="Binds Target strand DNA"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT SITE 823
FT /note="Binds Target strand DNA"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT SITE 826
FT /note="Binds Target strand DNA; via amide nitrogen"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT SITE 833
FT /note="Binds crRNA"
FT /evidence="ECO:0000269|PubMed:28431230"
FT SITE 852
FT /note="Stabilizes transition state for pre-crRNA
FT processing"
FT /evidence="ECO:0000305|PubMed:28431230"
FT SITE 1026
FT /note="Binds DNA in crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT SITE 1063
FT /note="Binds DNA in crRNA-target DNA heteroduplex"
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT MUTAGEN 608
FT /note="G->A,E: 15\% DNA cleavage."
FT /evidence="ECO:0000269|PubMed:28562584"
FT MUTAGEN 663
FT /note="P->A: 25\% DNA cleavage, altered non-target strand
FT cleavage products."
FT /evidence="ECO:0000269|PubMed:28562584"
FT MUTAGEN 666
FT /note="N->A: 80\% DNA cleavage, altered non-target strand
FT cleavage products."
FT /evidence="ECO:0000269|PubMed:28562584"
FT MUTAGEN 667
FT /note="K->A: 30\% DNA cleavage."
FT /evidence="ECO:0000269|PubMed:28562584"
FT MUTAGEN 671
FT /note="K->A: 15\% DNA cleavage."
FT /evidence="ECO:0000269|PubMed:28562584"
FT MUTAGEN 677
FT /note="K->A: 35\% DNA cleavage, altered non-target strand
FT cleavage products."
FT /evidence="ECO:0000269|PubMed:28562584"
FT MUTAGEN 692
FT /note="R->A: Slight decrease in target DNA cleavage, 30\%
FT DNA cleavage, altered non-target strand cleavage products."
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT MUTAGEN 694
FT /note="H->A: Wild-type DNA cleavage, altered non-target
FT strand cleavage products."
FT /evidence="ECO:0000269|PubMed:28562584"
FT MUTAGEN 698..702
FT /note="TKNGS->AGGGG: Loss of target DNA cleavage."
FT /evidence="ECO:0000269|PubMed:28431230"
FT MUTAGEN 704
FT /note="Q->A: Significant decrease in target DNA cleavage."
FT /evidence="ECO:0000269|PubMed:28431230"
FT MUTAGEN 843
FT /note="H->A: Decreased pre-crRNA processing in vitro, binds
FT RNA, no change in DNA cleavage."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 852
FT /note="K->A: Decreased pre-crRNA processing in vitro, binds
FT RNA, no change in DNA cleavage."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 869
FT /note="K->A: Decreased pre-crRNA processing in vitro, binds
FT RNA, no change in DNA cleavage."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 873
FT /note="F->A: Decreased pre-crRNA processing in vitro, no
FT pre-crRNA processing in E.coli, binds RNA, no change in DNA
FT cleavage."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 917
FT /note="D->A: Loss of target and non-target strand DNA
FT cleavage, no change in DNA-binding or pre-crRNA
FT processing."
FT /evidence="ECO:0000269|PubMed:26422227,
FT ECO:0000269|PubMed:27096362, ECO:0000269|PubMed:28431230"
FT MUTAGEN 920
FT /note="E->A: No longer cleaves DNA in presence of Ca(2+)."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 922
FT /note="H->A: Decreased cleavage of target strand in
FT presence of Ca(2+), wild-type cleavage of DNA in presence
FT of Mg(2+)."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 925
FT /note="Y->A: Decreased cleavage of target strand in
FT presence of Ca(2+), wild-type cleavage of DNA in presence
FT of Mg(2+)."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 1006
FT /note="E->A: Loss of target and non-target strand DNA
FT cleavage, no change in DNA-binding or pre-crRNA
FT processing."
FT /evidence="ECO:0000269|PubMed:26422227,
FT ECO:0000269|PubMed:27096362, ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT MUTAGEN 1006
FT /note="E->Q: Complete loss of DNA cleavage, still binds
FT crRNA; when associated with A-1218."
FT /evidence="ECO:0000269|PubMed:28431230"
FT MUTAGEN 1024
FT /note="Y->A: No longer cleaves DNA in presence of Ca(2+)."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 1028
FT /note="E->A: No longer cleaves DNA in presence of Ca(2+),
FT reduced cleavage of non-target strand in presence of
FT Mg(2+)."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 1065..1066
FT /note="KK->AA: 67\% DNA cleavage, altered non-target strand
FT cleavage products."
FT /evidence="ECO:0000269|PubMed:28562584"
FT MUTAGEN 1218
FT /note="R->A: Cleaves both target and non-target strand DNA.
FT Complete loss of DNA cleavage, still binds crRNA; when
FT associated with Q-1006."
FT /evidence="ECO:0000269|PubMed:28431230,
FT ECO:0000269|PubMed:28562584"
FT MUTAGEN 1227
FT /note="D->A: No longer cleaves DNA in presence of Ca(2+)."
FT /evidence="ECO:0000269|PubMed:27096362"
FT MUTAGEN 1255
FT /note="D->A: Significant reduction to loss of target and
FT non-target strand DNA cleavage, no change in DNA-binding or
FT pre-crRNA processing."
FT /evidence="ECO:0000269|PubMed:26422227,
FT ECO:0000269|PubMed:27096362, ECO:0000269|PubMed:28431230"
FT MUTAGEN 1255
FT /note="D->N: Significant reduction of target and non-target
FT strand DNA cleavage."
FT /evidence="ECO:0000269|PubMed:28431230"
FT TURN 3..6
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 13..23
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 27..34
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 36..68
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 73..86
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 92..114
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 117..120
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 122..124
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 125..127
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 132..134
FT /evidence="ECO:0007829|PDB:6I1L"
FT HELIX 137..147
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 153..155
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 157..159
FT /evidence="ECO:0007829|PDB:6I1K"
FT HELIX 162..171
FT /evidence="ECO:0007829|PDB:5NFV"
FT TURN 172..174
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 176..179
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 180..190
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 192..194
FT /evidence="ECO:0007829|PDB:6I1K"
FT STRAND 196..198
FT /evidence="ECO:0007829|PDB:5NG6"
FT HELIX 199..204
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 207..224
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 226..228
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 231..237
FT /evidence="ECO:0007829|PDB:5NFV"
FT TURN 238..242
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 243..245
FT /evidence="ECO:0007829|PDB:5NFV"
FT TURN 248..251
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 252..257
FT /evidence="ECO:0007829|PDB:6I1L"
FT HELIX 260..263
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 267..271
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 272..274
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 275..286
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 291..293
FT /evidence="ECO:0007829|PDB:5MGA"
FT STRAND 294..297
FT /evidence="ECO:0007829|PDB:5NG6"
FT HELIX 300..311
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 314..319
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 345..361
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 365..367
FT /evidence="ECO:0007829|PDB:6I1K"
FT HELIX 370..382
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 388..390
FT /evidence="ECO:0007829|PDB:5NG6"
FT STRAND 392..394
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 397..406
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 412..422
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 424..426
FT /evidence="ECO:0007829|PDB:6GTG"
FT STRAND 427..432
FT /evidence="ECO:0007829|PDB:6I1L"
FT HELIX 435..445
FT /evidence="ECO:0007829|PDB:6I1K"
FT STRAND 449..452
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 453..465
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 469..471
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 475..484
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 487..506
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 508..510
FT /evidence="ECO:0007829|PDB:6I1L"
FT HELIX 513..515
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 517..519
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 520..541
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 559..572
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 575..586
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 595..597
FT /evidence="ECO:0007829|PDB:5NFV"
FT TURN 603..606
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 611..613
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 614..617
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 619..624
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 627..633
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 635..637
FT /evidence="ECO:0007829|PDB:6I1K"
FT TURN 638..641
FT /evidence="ECO:0007829|PDB:6I1K"
FT HELIX 643..648
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 650..661
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 665..673
FT /evidence="ECO:0007829|PDB:5NFV"
FT TURN 676..678
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 679..682
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 686..694
FT /evidence="ECO:0007829|PDB:5NFV"
FT TURN 695..697
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 698..700
FT /evidence="ECO:0007829|PDB:6I1L"
FT HELIX 704..706
FT /evidence="ECO:0007829|PDB:6I1L"
FT HELIX 714..730
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 731..733
FT /evidence="ECO:0007829|PDB:6I1K"
FT HELIX 734..737
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 744..746
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 750..760
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 761..769
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 771..779
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 782..789
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 791..793
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 803..812
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 814..818
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 821..824
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 829..833
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 845..847
FT /evidence="ECO:0007829|PDB:6GTG"
FT STRAND 852..856
FT /evidence="ECO:0007829|PDB:5MGA"
FT STRAND 857..861
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 871..874
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 877..887
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 888..890
FT /evidence="ECO:0007829|PDB:6GTG"
FT HELIX 896..906
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 908..910
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 912..917
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 919..921
FT /evidence="ECO:0007829|PDB:6GTG"
FT STRAND 923..929
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 931..933
FT /evidence="ECO:0007829|PDB:6I1K"
FT STRAND 935..944
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 947..952
FT /evidence="ECO:0007829|PDB:6I1K"
FT HELIX 953..968
FT /evidence="ECO:0007829|PDB:5NFV"
FT TURN 969..971
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 977..999
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1001..1006
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1008..1011
FT /evidence="ECO:0007829|PDB:6I1K"
FT HELIX 1016..1018
FT /evidence="ECO:0007829|PDB:6GTG"
FT HELIX 1019..1036
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1041..1043
FT /evidence="ECO:0007829|PDB:5MGA"
FT STRAND 1045..1047
FT /evidence="ECO:0007829|PDB:6I1K"
FT STRAND 1050..1053
FT /evidence="ECO:0007829|PDB:6I1K"
FT HELIX 1065..1067
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1069..1071
FT /evidence="ECO:0007829|PDB:6I1L"
FT STRAND 1074..1077
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1083..1085
FT /evidence="ECO:0007829|PDB:5NFV"
FT TURN 1087..1089
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 1102..1110
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1112..1118
FT /evidence="ECO:0007829|PDB:5NFV"
FT TURN 1119..1122
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1123..1129
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 1130..1132
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1133..1135
FT /evidence="ECO:0007829|PDB:6GTG"
FT STRAND 1136..1138
FT /evidence="ECO:0007829|PDB:6I1L"
FT STRAND 1141..1145
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1150..1153
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1157..1162
FT /evidence="ECO:0007829|PDB:6GTG"
FT STRAND 1165..1168
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 1170..1180
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 1192..1197
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 1201..1214
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1218..1220
FT /evidence="ECO:0007829|PDB:6I1L"
FT TURN 1222..1225
FT /evidence="ECO:0007829|PDB:5NG6"
FT STRAND 1228..1230
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1236..1238
FT /evidence="ECO:0007829|PDB:6I1K"
FT STRAND 1242..1245
FT /evidence="ECO:0007829|PDB:5NFV"
FT HELIX 1254..1275
FT /evidence="ECO:0007829|PDB:5NFV"
FT STRAND 1278..1280
FT /evidence="ECO:0007829|PDB:6I1K"
FT HELIX 1288..1297
FT /evidence="ECO:0007829|PDB:5NFV"
SQ SEQUENCE 1300 AA; 151915 MW; 601E903DE68C80DE CRC64;
ALIGNMENT:
Query Match 100.0%; Score 6763; Length 1300;
Best Local Similarity 100.0%;
Matches 1300; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF 60
Qy 61 FIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFK 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 FIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFK 120
Qy 121 NLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFK 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 NLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFK 180
Qy 181 GFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE 240
Qy 241 ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI 300
Qy 301 NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIA 360
Qy 361 AFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 AFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 420
Qy 421 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA 480
Qy 481 NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKL 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKL 540
Qy 541 KIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 KIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF 600
Qy 601 ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYK 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYK 660
Qy 661 LLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 LLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF 720
Qy 721 IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQ 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQ 780
Qy 781 GKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 GKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 840
Qy 841 ITHPAKEAIA NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 ITHPAKEAIA NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI 900
Qy 901 NLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 NLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI 960
Qy 961 EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVE 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVE 1020
Qy 1021 KQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAG 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 KQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAG 1080
Qy 1081 FTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKG 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 FTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKG 1140
Qy 1141 KWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 KWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD 1200
Qy 1201 KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAY 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAY 1260
Qy 1261 HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 1300
||||||||||||||||||||||||||||||||||||||||
Db 1261 HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 1300
Miao et al. teach DNase deactivated F. novicida Cpf1 variants, including variants of the F. novicida Cpf1 that have a single or double substitution, namely substitutions D917A and/or E1006A (page 2, right column, last 10 lines-page 3, left column). The substitution D917A corresponds to the substitution D832A in the polypeptide of SEQ ID NO: 4 according to Miao et al. (page 3, left column, line 27). Miao et al. teach the use of dCpf1 as DNA binding domains in fusion proteins that act as transcriptional regulators, the advantages of Cpf1 due to the fact that it does not require tracrRNAs as it can process crRNAs co-transcripts into independent mature crRNAs (page 1, right column), and the fact that it has a thymine-rich PAM preference that extends the targetable regions especially in AT-rich genomes (page 2, left column, lines 1-19). Neither Safari et al. nor Miao et al. teach a Spo11 protein or variant thereof.
With regard to the argument that neither Bastianelli et al., Safari et al. nor Miao et al. teach or suggest the possibility of using the CRISPR/Cpf1 system to induce recombination during meiosis of a eukaryotic cell, it is noted that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, the Examiner has previously indicated that a person of ordinary skill in the art would have been motivated to replace the dCas9 domain of the fusion protein of Bastianelli et al. with the dCpf1 protein of Miao et al. or Safari et al. for the benefit of targeting AT-rich DNA targets in the genome that cannot be reached by the dCas9 domain of the fusion protein of Bastianelli et al., and the use of a concise guide RNA (crRNA) which is more compatible with current DNA oligomer synthesis techniques, as taught by Miao et al. As taught by Safari et al. and Miao et al., dCpf1 has been used as a DNA binding domain in other fusion proteins.
With regard to the argument that (i) during meiosis, DNA takes on vary different forms and goes through different processes, (ii) cellular physiology is also unique during meiosis, with variations in iron content and intracellular pH, (iii) the cellular conditions during meiosis could impact the stability and activity of the Cpf1 complex by leading to changes in the structure of the gRNA and/or Cpf1 protein that are necessary for targeting and DNA binding, (iv) specific RNA binding proteins expressed during meiosis could interfere with the guide RNA, and (v) methylation and compaction can have the ability to negatively impact DNA targeting and binding, it is noted that while it is agreed that several changes occur during meiosis, there is no indication or evidence to suggest that these changes would affect Cpf1 and its guide RNA and not affect Cas9 and its guide RNA. For example, there is no indication that the pH and iron content present during meiosis would not affect Cas9 but would affect Cpf1. It should also be noted that even if the argument is made that these changes in pH and iron content would affect Cpf1 enzymatic activity, the fusion protein of Bastianelli et al. comprises a catalytically inactive Cas protein (Cas9) and the fusion protein of the claims comprises a catalytically inactive Cas protein (Cpf1 or Cas12a). There is no evidence to suggest that RNA binding proteins expressed during meiosis that did not interference with the Cas9 guide RNA would interfere with the guide RNA of a Cpf1 protein. Both Cas9 and Cpf1 are proteins which are endonucleases and are over 1000 amino acids long. Moreover, even if the argument is made that the endogenous guide RNA of a Cas9 is not structurally similar to that of a Cpf1 protein, it is noted that single guide RNAs for Cas9 are not only well known in the art but are used in the method of Bastianelli et al. Therefore, the guide RNA used by Bastianelli et al. is a single guide RNA, which is also the case for a Cpf1 guide RNA. Similarly, there is no evidence to show that methylation and compaction would negatively impact DNA targeting and binding if a Cpf1 system is used but do not negatively impact DNA targeting and binding if a Cas9 system is used.
With regard to the argument that because (i) Cpf1 produces a double strand break with sticky ends while Cas9 produces clean ends after cleavage, and (ii) the guide RNA of a Cpf1 is not the same as that of a Cas9 because a Cpf1 guide RNA does not require a tracrRNA, one of skill in the art would not substituting a Cas9 protein with a Cpf1 protein in the context of a meiotic cell, it is noted that the fusion proteins of Bastianelli et al. can make double strand breaks via the Spo11 domain. Bastianelli et al. teach that the Spo11 domain of the Cas9-Spo11 fusion protein is generally the domain responsible for double strand breaks (page 5, paragraph [0092]). It is reiterated herein that the guide RNA used by Bastieanelli et al. is a single guide RNA and not one that comprises two separate structures (crRNA and tracrRNA). Therefore, whether the Cpf1 and Cas9 proteins make different types of double strand breaks does not appear to be an issue in the instant case if the Spo11 domain is the one responsible for the double strand breaks.
With regard to the argument that the specification provides unexpected results, it is noted that it appears that it would not be unexpected to observe the induction of DSBs and meiotic recombination if the part of the fusion protein that is responsible for double strand breaks is the same (i.e., Spo11) in the protein of Bastianelli et al. and fusion protein of Bastianelli et al., Safari et al. and Miao et al. Therefore, for the reasons of record and those set forth above, one of skill in the art would conclude that the claimed invention is obvious over the cited prior art.
Double Patenting
Claim 41 was provisionally rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claim 40 of copending Application No. 17/926,208. In view of the amendment of claim 40 in copending Application No. 17/926,208, which now requires a class II type II CRISPR nuclease that has deficient nuclease activity, this rejection is hereby withdrawn.
Conclusion
No claim is in condition for allowance.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a). A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
Applicant is advised that any Internet email communication by the Examiner has to be authorized by Applicant in written form. See MPEP § 502.03 (II). Without a written authorization by Applicant in place, the USPTO will not respond via Internet email to any Internet correspondence which contains information subject to the confidentiality requirement as set forth in 35 U.S.C. 122. Sample written authorization language can be found in MPEP § 502.03 (II). An Authorization for Internet Communications in a Patent Application or Request to Withdraw Authorization for Internet Communications form (SB/439) can be found at https://www.uspto.gov/patent/forms/ forms-patent-applications-filed-or-after-september-16-2012, which can be electronically filed.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DELIA M RAMIREZ, Ph.D., whose telephone number is (571) 272-0938. The examiner can normally be reached on Monday-Friday from 8:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert B. Mondesi, can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
/DELIA M RAMIREZ/Primary Examiner, Art Unit 1652
DR
May 26, 2026
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