EENdb - Utilities  >  FokI variants
Summary
The variants of FokI cleavage domains used in constructing EENs can be classified to some groups according to the purpose of mutations induced into the wild-type variant.
  1. Hetero-dimer mutants: FokI variants with mutations on the interface of dimerization to reduce the efficiency of homodimers of wild-type FokI. The specificities of EENs (especially those of ZFNs) with these variant pairs are reported to be improved.
  2. Activity enhanced mutants: FokI variants with specific mutations to enhance the activity, named as "Sharkey" variants.
  3. Nickase mutations: FokI variants with mutations in the catalytic activity center of one domain of the pair to disrupt its cleavage activity, which causes the pair inactive as a nuclease but active as a nickase, i.e., only cut one strand of DNA and leave a nick. The nicks are reported to largely reduce the efficiency of NHEJ with less affecting the efficiency of HR.
  4. Cold-sensitive mutants: FokI variants with specific mutations that the cleavage activity is diminished at lower temperatures.
These types of mutations can be employed in one variant.

Click on the variant name to see its sequence; click here to see the map of candidate mutation site.
Classif. Variants Mutations Reference Note
Wild type WT (none) 8577732
EENs with WT
Last 196 aa (384-579) of the FokI endonuclease.
The numbers of the residues are actually 388-583 in Uniprot. However, all the variants shown below do not count the first 4 aa of the peptide customarily by the references.
I: Hetero-dimer EL + KK EL: Q486E, I499L
KK: E490K, I538K
17603475
EENs with EL+KK
D + R D: R487D
R: D483R
17603476
EENs with D+R
Most articles call these variants as "FokI-DD" and "FokI-RR". However, "FokI-D" and "FokI-R" are recommended for these two single-mutated variants by 21131970 (FokI-DD and FokI-RR are referred to other double-mutated variants).
EA + KV EA: Q486E, I499A
KV: E490K, I538V
17603476
EENs with EA+KV
A + V
DA + RV
A: I499A, V: I538V
D: R487D, R: D483R
17603476 (Only constructed EENs targeting reported sites.)
REL + DKK
RELV + DKKA
R-EL, D-KK, R-EL-V, D-KK-A:
(see R+D, EL+KK and A+V)
21094162 (Only constructed EENs targeting reported sites.)
ELD + KKR ELD: Q486E, I499L, N496D
KKR: E490K, I538K, H537R
21131970
EENs with ELD+KKR
ELD + KKK
ELE+KKK
ELE+KKR
DAD+RVK
DAD+RVR
DD+RR
ELD: Q486E, I499L, N496D
ELE: Q486E, I499L, N496E
KKK: E490K, I538K, H537K
KKR: E490K, I538K, H537R
DD: R487D, N496D
DAD: R487D, I499A, N496D
RR: D483R, H537R
RVR: D483R, I538V, H537R
21131970 (Only constructed EENs targeting reported sites.)
Most articles call the variants "FokI-D" and "FokI-R" as "FokI-DD" and "FokI-RR". However, they are not the variants "FokI-DD" and "FokI-RR" listed here.
II: Activity enhanced Sharkey S418P, K441E 20447404
EENs with Sharkey
Sharkey variants were reported to show higher activity than the WT ones.
Sharkey' S418P, F432L, K441E, Q481H, H523Y, N527D, K559Q 20447404 (Only constructed EENs targeting reported sites.)
(I + II) Sharkey-AS + -R (see Sharkey' and R in D+R)
-AS: E484A, R487S
20447404 These variants are called "FokI-Sharkey-DAMQS"/"FokI-Sharkey-DS" and "FokI-Sharkey-RR" in the reference. EENdb renamed them according to the nomenclature guideline by 21131970.
Sharkey'-D + -R
Sharkey'-AS + -R
Sharkey'-EL + -KK
(see Sharkey' and D+R, EL+KK)
-AS: E484A, R487S
20447404 The first three variants are called "FokI-Sharkey'-DS", "FokI-Sharkey'-DD" and "FokI-Sharkey'-RR" in the reference. EENdb renamed them according to the nomenclature guideline by 21131970.
Sharkey-ELD + -KKR (see Sharkey and ELD+KKR) 21131970
Sharkey-EL + -KK
Sharkey-DA + -RV
Sharkey-DAD + -RVR
(see Sharkey and EL+KK, DA+RV, DAD+RVR) 21131970 (Only constructed EENs targeting reported sites.)
III: Nickase * D450A (in one domain) 22373919
22522391
(unnamed) D450N (in one domain) or
D467A (in one domain)
22434427 D467A is reported to mainly show nickase activity but with low remaining nuclease activity.
(I + III) EL* + KK
EL + KK*
(see EL+KK and *) 22373919
22522391
The nickase activities of the dimer are reported to be affected by which variant carry the nickase mutation.
IV: Cold-sensitive (various) (various) 21131970 For more details, see the reference.
Non-FokI Snase (another non-specific endonuclease domain) 18980382 Used in some "Sandwich" ZFNs.
FokI-StsI-RE + -DKK FokI-StsI: (fusion domain)
RE: D489R, G492E
DKK: R493D, R496K, D538K
21094162 In FokI-StsI variants, residues 485-496, 527-539 of FokI are replaced by StsI.
(Only constructed EENs targeting reported sites.)


Candidate mutation sites in WT FokI sequence
WT Type of candidate mutation sites: I II III 390 400 410 420 ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ S418P^ -- Sharkey (1 of 2) 430 440 450 460 470 480 DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG | ^K441E | | -- Sharkey (2 of 2) ^F432L | | -- Sharkey' (3 of 7) ^D450A/N ^D467A -- *,/(other nickase) 490 500 510 520 530 540 QADEMQRYVE ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN | || ^Q486E | ^I499L/A | | || -- EL/EA, A | || | ^E490K | | | I538K/V^ -- KK/KV, V E484A^ ^R487D/S ^N496D/E | | | -- AS/D(1st), D/E(2nd) | ^D483R | | H537R/K^ -- R(1st), R/K(2nd) ^Q481H H523Y^ ^N527D -- Sharkey' (4-6 of 7) 550 560 570 579 CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF ^K559Q -- Sharkey' (7 of 7) e.g. DKKA + RELV = D(1st)-KK-A + R(1st)-EL-V = R487D E490K I538K I499A + D483R Q486E I499L I538V ELD + KKR = EL-D(2nd) + KK-R(2nd) = Q486E I499L N496D + E490K I538K H537R DD + RR = D(1st)-D(2nd) + R(1st)-R(2nd) = R487D N496D + D483R H537R Sharkey-AS + Sharkey-R = Sharkey-AS + Sharkey-R(1st) = S418P K441E E484A R487S + S418P K441E D483R EL* + KK = EL-* + KK = Q486E I499L D450A + E490K I538K




WT FokI nuclease domain
Reference   List EENs with WT FokI

WT ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMQRYVE ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF

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FokI EL+KK
Reference   List EENs with EL+KK FokI

FokI-EL: Q486E I499L ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMERYVE ENQTRNKHLN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF FokI-KK: E490K I538K ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMQRYVK ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHKTN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF

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FokI D+R
Reference   List EENs with D+R FokI

FokI-D: R487D ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMQDYVE ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF FokI-R: D483R ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QAREMQRYVE ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF

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FokI EA+KV
Reference   List EENs with EA+KV FokI

FokI-EA: Q486E I499A ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMERYVE ENQTRNKHAN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF FokI-KV: E490K I538V ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMQRYVK ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHVTN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF

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FokI ELD+KKR
Reference   List EENs with ELD+KKR FokI

FokI-ELD: Q486E I499L N496D ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMERYVE ENQTRDKHLN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF FokI-KKR: E490K I538K H537R ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNSTQ DRILEMKVME FFMKVYGYRG KHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMQRYVK ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNRKTN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF

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FokI Sharkey-AS + Sharkey-R
Reference   List EENs with Sharkey FokI

FokI-Sharkey-AS: S418P K441E E484A R487S ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNPTQ DRILEMKVME FFMKVYGYRG EHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADAMQSYVE ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF FokI-Sharkey-R: S418P K441E D483R ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNPTQ DRILEMKVME FFMKVYGYRG EHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QAREMQRYVE ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF

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FokI Sharkey-ELD + Sharkey-KKR
Reference   List EENs with Sharkey FokI   List EENs with ELD+KKR FokI

FokI-Sharkey-ELD: S418P K441E Q486E I499L N496D ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNPTQ DRILEMKVME FFMKVYGYRG EHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMERYVE ENQTRDKHLN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNHITN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF FokI-Sharkey-KKR: S418P K441E E490K I538K H537R ... QLVKSEL EEKKSELRHK LKYVPHEYIE LIEIARNPTQ DRILEMKVME FFMKVYGYRG EHLGGSRKPD GAIYTVGSPI DYGVIVDTKA YSGGYNLPIG QADEMQRYVK ENQTRNKHIN PNEWWKVYPS SVTEFKFLFV SGHFKGNYKA QLTRLNRKTN CNGAVLSVEE LLIGGEMIKA GTLTLEEVRR KFNNGEINF

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