SUPPLEMENTARY INFORMATION
Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genom
e Engineering Applications
Mustapha Aouida, Ayman Eid, Zahir Ali, Thomas Cradick, Ciaran Lee, Harshavardhan Deshmu
kh, Ahmed Atef, Dina AbuSamra, , Samah Zeineb Gadhoum, Jasmeen Merzaban, Gang Bao and
Magdy M. Mahfouz*
*Author for correspondence: Magdy Mahfouz (Magdy.mahfouz@kaust.edu.sa)
Supplementary Fig. A
T7EI assays at EMX1, AAVS1, CCR5, and HBB tar
gets with wtCas9
Supplementary Fig. B
Target genome modification of EMX1, AAVS1, C
CR5, and HBB using wtCas9 and various gRNAs
Supplementary Fig. C
T7EI assays at EMX1, AAVS1, CCR5, and HBB wi
th fdCas9 using a single gRNA
Supplementary Fig. D
T7EI assays at EMX1, AAVS, CCR5, and HBB targ
ets with dCas9, Cas9 nickases, and dCas9f and
a pair of gRNAs in PAM-in and PAM-out orienta
tions.
Supplementary Fig. E
Target genome modification of EMX1, AAVS1, C
CR5, and HBB using Cas9 nickases and a pair of
gRNAs
Supplementary Fig. F
Comparison of the genome editing activities of
fdCas9 and the Cas9 nickases with a pair of gR
NAs in the PAM-out orientation.
Supplementary Fig. G
Target genome modification of HBD and CCR2
using wtCas9 and a pair of gRNAs for HBB and
CCR5, respectively
Supplementary Fig. H
T7EI assays in HBD and CCR2 targets using Cas
9 nickases
Supplementary Fig. I
Sharkey FokI variant displayed same activity a
s the wt.FokI
Supplementary Tables
Table A; B; C; D; E; F; G; H
Additional Supplementary informat pMRS reporter system, gRNA combinations of t
ion
argets, Constructs, DNA and amino acid sequen
cing results for dCas9, fdCas9, and dCas9f varia
nts
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Table A: Primers used for cloning and sequencing of fdCas9, dCas9f, and different spacers f
or pMRS.
Name
Primer Sequence
dCas9H.MluI.F
CCGAGTTTTCTAAACGCGTCATTCTCGCTGATGC
dCas9H.RI.X.ApI.R
GCGGAATTCTCGAGGGGCCCTACCTTTCTCTTCTT
TTTTGGATCTACC
FokI.FApaI
dCas9H. F1
ACGGGGCCCAAGCAACTAGTCAAAAGTGAACTGGA
GGAGAAG
GCGGAATTCTCGAGTCAAAAGTTTATCTCGCCGTT
ATTA
GTAATGAGCTGGCTCTCCCCTCCAAGTACGTG
dCas9H. F2
CCCTCATCCACCAGAGCATCACCGGACTTTAC
dCas9H. R1
GTACTTGAATGCGGCAGGGGCACCAAGATTGG
dCas9H. FokI.R2
CCTCTATATCCATAAACTTTCATAAAAAATTC
pMRS.seqcF1
GCTGGACATCACCTCCCACAACGAGGACTACACCA
T
GAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAG
FokI.R.Xh1.R1
pMRS.seqcR1
19
Used
Amplify human dCas9 fragme
nt MluI XhoI to remove the co
don stop from dCas9
Amplify human dCas9 fragme
nt MluI XhoI to remove the co
don stop from dCas9
Amplify FokI to be inserted in
human dCas9
Amplify FokI to be inserted in
human dCas9
To verify the fusion of human
dCas9-FoKI
To verify the fusion of human
dCas9-FoKI
To verify the fusion of human
dCas9-FoKI
To verify the fusion of human
dCas9-FoKI
To verify the spacer cloning
To verify the spacer cloning
Primers with restriction EcoRI and BamHI sequence over-hangs used for spacer cloni
ng:
Table B: Primer sequences for spacer cloning in the pMRS reporter plasmid.
Primers names
pMRS-sp3-F
pMRS-sp3-R
pMRS-sp6-F
pMRS-sp6-R
pMRS-sp9-F
pMRS-sp9-F
pMRS-sp12-F
pMRS-sp12-R
pMRS-sp15-F
pMRS-sp15-R
pMRS-sp18-F
pMRS-sp18-R
pMRS-sp21-F
pMRS-sp21-R
pMRS-sp24-F
pMRS-sp24-R
pMRS-sp27-F
pMRS-sp27-R
pMRS-sp30-F
pMRS-sp30-R
pMRS-sp33-F
pMRS-sp33-R
pMRS-sp36-F
pMRS-sp36-R
pMRS-sp39-F
pMRS-sp39-R
sequence 5'-……- 3' (EcoRI-…….- BamHI)
aattcCCGCTAGTGCGATGGCCTCATGGAAGCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCAGCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGCTGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCAGCCACCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGTGGCTGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCAGCCAGGACCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGTCCTGGCTGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCAGCCAGGACAACCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGTTGTCCTGGCTGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCAGCCAGGACAATTTCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGAAATTGTCCTGGCTGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCAGCCAGGACAATTTACCCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGGGTAAATTGTCCTGGCTGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCAGCCAGGACAATTTACCGATCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGATCGGTAAATTGTCCTGGCTGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCAGCCAGGACAATTTACCGATCATCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGATGATCGGTAAATTGTCCTGGCTGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
aattcCCGCTAGTGCGATGGCCTCATGGAAGCTTGATATCCAGCCAGGACAATTTACCGATCATtCGCCCATGAGGCCATCGCACTAGGGGg
gatccCCCCTAGTGCGATGGCCTCATGGGCGATGATCGGTAAATTGTCCTGGCTGGATATCAAGCTTCCATGAGGCCATCGCACTAGCGGg
Table C: Human gRNA target sequences for EMX1.
gRNA Name
gEMX1-1
gEMX1-2
gEMX1-3
gEMX1-4
gEMX1-5
gEMX1-6
gEMX1-7
gEMX1-14
gEMX1-16
20
gRNA Sequence (5′-………-3′)
GAGTCCGAGCAGAAGAAGAA
AGGGCTCCCATCACATCAAC
CACGAAGCAGGCCAATGGGG
GCGCCACCGGTTGATGTGAT
GCTTCGTGGCAATGCGCCAC
CTCCCCATTGGCCTGCTTCG
GACATCGATGTCCTCCCCAT
TCACCTCCAATGACTAGGGT
GATGTCACCTCCAATGACTA
Table D: Human gRNA target sequences for AAVS1.
gRNA Name
gAAVS1-1
gAAVS1-2
gAAVS1-3
gAAVS1-4
gAAVS1-10
gAAVS1-11
gRNA Sequence (5′-………-3′)
GGATCCTGTGTCCCCGAGCT
GAGCCACATTAACCGGCCCT
GTTAATGTGGCTCTGGTTCT
GGCCCCACTGTGGGGTGGAG
TGTCCCCTCCACCCCACAGT
AGAACCAGAGCCACATTAAC
Table E: Human gRNA target sequences for CCR5.
gRNA Name
gCCR5-1
gCCR5-2
gCCR5-3
gCCR5-4
gCCR5-5
gCCR5-6
gCCR5-7
gCCR5-11
gCCR5-12
gCCR5-13
gCCR5-14
gCCR5-15
gCCR5-16
gRNA Sequence (5′-………-3′)
CCTTCTTACTGTCCCCTTCT
ATTTCCAAAGTCCCACTGGG
TGTATTTCCAAAGTCCCACT
TGGTTTTGTGGGCAACATGC
TTTTGCAGTTTATCAGGATG
TCAGCCTTTTGCAGTTTATC
GTGTTCATCTTTGGTTTTGT
TGACATCTACCTGCTCAACC
CCCAGAAGGGGACAGTAAGA
GGACAGTAAGAAGGAAAAAC
CAGCATAGTGAGCCCAGAAG
GCTGCCGCCCAGTGGGACTT
CAATGTGTCAACTCTTGACA
Table F: Human gRNA target sequences for HBB.
gRNA Name
gHBB-1
gHBB-2
gHBB-3
gHBB-4
gHBB-5
gHBB-8
gHBB-9
gHBB-10
21
gRNA Sequence (5′-………-3′)
CCTGTGGGGCAAGGTGAACG
CCTTGATACCAACCTGCCCA
AAGGTGAACGTGGATGAAGT
GTGAACGTGGATGAAGTTGG
GTGAACGTGGATGAAGTTGG
CACGTTCACCTTGCCCCACA
CCCTGGGCAGGTTGGTATCA
CTTGCCCCACAGGGCAGTAA
gHBB-11
gHBB-12
GAAGTTGGTGGTGAGGCCCT
TTGGTGGTGAGGCCCTGGGC
Table G: Primers used to amplify the U6.gRNAs of AAVS1, EMX1, HBB, and CCR5 from hgRN
A plasmids for direct transfection of PCR products.
Name
Primer Sequence
Used
U6-EMX1gRNA-F
GGGCCCGCTCTAGAGATCCGACGCGCCATC
U6-EMX1gRNA-R
AGTTATGTAACGCGGAACTCCATATATGGG
Amplify hgRNA by
PCR
Amplify hgRNA by
PCR
U6-AAVS1gRNA-F
U6-AAVS1gRNA-R
U6-HBBgRNA-F
U6-HBBgRNA-R
U6-CCR5gRNA-F
U6-CCR5gRNA-F
GGGCCCGCTCTAGAGATCCGACGCGCCATC
AGTTATGTAACGCGGAACTCCATATATGGG
GCGGCCGCGGGCCCGCTCTAGAGAT
GGATCCTAGTACTCGAGAAAAAAAG
GCGGCCGCGGGCCCGCTCTAGAGAT
GGATCCTAGTACTCGAGAAAAAAAG
Table H: Primers used to amplify EMX1, AAVS1, HBB, CCR5, HBD, and CCR2 from human ge
nomic DNA for the T7EI endonuclease assay.
gRNA Name
Primer Sequence (5′-….-3′)
EMX1-F
EMX1-R
AAVS1-F
AAVS1-R
AAVS1-2F
AAVS1-2R
HBB-F
HBB-R
CCR5-F
CCR5-R
HBD-F
HBD-R
CCR2-F
CCR2-R
GAACAGGAAAACCACCCTTCTCTC
CAGCCAGCCCATTGCTTGTC
TAGTCTTCTTCCTCCAACCCGGGCCCCT
TCCAGGAAATGGGGGTGTGTCACCAGA
TTTAGCCACCTCTCCATCCTCTTG
TGGCTACTGGCCTTATCTCACAGG
AGCCAGGGCTGGGCATAAAAG
CCAATAGGCAGAGAGAGTCAGTG
GACATCAATTATTATACATCGGAGC
GACGCAAACACAGCCACCACCCAAG
GTGATGGCCTGGCTCACCTGGACAACCTC
TACATATCTCCCCACCGCATCTCTTTCAGCAG
TTGAACAAGGACGCATTTCCCCAG
CAAAGACCCACTCATTTGCAGCAG
22
Amplicon size (b
p)
590
590
301
301
842
842
298
298
449
449
653
653
361
361
Fragment sequences for the N-terminal fusion
FLAG-NLS-WT.FOKI-Linker-dCas9:
CCCTTCACCATGGATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACA
AGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACC
TGGAGGTTCTATGGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGT
CATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTC
AGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAA
ACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTAC
GGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATG
AAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTG
GAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGA
AACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTG
TAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTCAG
ACGGAAATTTAATAACGGCGAGATAAACTTTAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCG
CCACACCCGAAACCCCATGGCCCTTCA
FLAG-NLS-Sharky.FOKI-Linker-dCas9:
CCCTTCACCATGGATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACA
AGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACC
TGGAGGTTCTATGGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGT
CATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATCCCACTC
AGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTGA
ACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTAC
GGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATG
AAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTG
GAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGA
AACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTG
TAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAG
ACGGAAATTTAATAACGGCGAGATAAACTTT
I23
Spacers with PAM-in orientation cloned in pMRS plasmid
pMRS-gRNA PAM-out (F and R): 5′-GAGTCCGAGCAGAAGAAGAA-3′
Spacer 2:
Spacer 5:
Spacer 8:
Spacer 11:
Spacer 14:
Spacer 17:
Spacer 20:
24
Spacer 23:
Spacer 26:
Spacer 29
Spacer 32:
Spacer 35:
Spacer 38:
25
II-
Spacers with PAM-out orientation cloned in pMRS plasmid
pMRS-gRNA PAM-out (F and R): 5′-CCATGAGGCCATCGCACTAG-3′
Spacer 3:
Spacer 6:
Spacer 9:
Spacer 12:
Spacer 15:
Spacer 18:
26
27
Spacer 21:
Spacer 24:
Spacer 27:
Spacer 30:
Spacer 33:
Spacer 36:
28
29
Spacer 39:
pMRS reporter plasmid used to clone the spacers with targets in the in and out orientations
to test the activity of the C- and N-terminus dCas9 and FokI fusions.
The pMRS reporter system and spacer size ranging from 3–39 bp.
30
I-
EMX1 target sequence:
GAACAGGAAAACCACCCTTCTCTCTGGCCCACTGTGTCCTCTTCCTGCCCTGCCATCCCCTTCTGTGAATGTTAGACC
CATGGGAGCAGCTGGTCAGAGGGGACCCCGGCCTGGGGCCCCTAACCCTATGTAGCCTCAGTCTTCCCATCAGGC
TCTCAGCTCAGCCTGAGTGTTGAGGCCCCAGTGGCTGCTCTGGGGGCCTCCTGAGTTTCTCATCTGTGCCCCTCCCT
CCCTGGCCCAGGTGAAGGTGTGGTTCCAGAACCGGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGG
GCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATG
GGGAGGACATCGATGTCACCTCCAATGACTAGGGTGGGCAACCACAAACCCACGAGGGCAGAGTGCTGCTTGCT
GCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAGTC
ATGGCCCCACAGGGCTTGAAGCCCGGGGCCGCCATTGACAGAGGGACAAGCAATGGGCTGGCTG
1- Different target sequences with PAM-in and PAM-out orientations.
2- Possible combinations of pairs of EMX1 gRNAs in PAM-in and PAM-out orientations:
31
II-
AAVS1 target sequence
TAGTCTTCTTCCTCCAACCCGGGCCCCTATGTCCACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTCC
CCGAGCTGGGACCACCTTATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCA
CCCCACAGTGGGGCCACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCCTTCCTAGTCTCC
TGATATTGGGTCTAACCCCCACCTCCTGTTAGGCAGATTCCTTATCTGGTGACACACCCCCATTTCCTGG
1- Different targets sequences with PAM-in and PAM-out orientations.
2- Possible combination of a pair of AAVS1 gRNAs in PAM -in and PAM-out from the
spacer:
32
33
III-
CCR5 target sequence
GACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCT
CCTGCCTCCGCTCTACTCACTGGTGTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCA
TCCTGATAAACTGCAAAAGGCTGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCT
GACCTGTTTTTCCTTCTTACTGTCCCCTTCTGGGCTCACTATGCTGCCGCCCAGTGGGACTTTGGA
AATACAATGTGTCAACTCTTGACAGGGCTCTATTTTATAGGCTTCTTCTCTGGAATCTTCTTCAT
CATCCTCCTGACAATCGATAGGTACCTGGCTGTCGTCCATGCTGTGTTTGCTTTAAAAGCCAGGA
CGGTCACCTTTGGGGTGGTGACAAGTGTGATCACTTGGGTGGTGGCTGTGTTTGC
1- Different targets sequences with PAM-in and PAM-out orientations.
2- Possible combinations of pairs of EMX1 gRNAs in PAM-in and PAM-out orientations:
34
35
IV-
HBB target sequence:
AGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAAC
TGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCACCTGACTCCTGAGGAGAAGTCTGCCGT
TACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTA
TCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACT
CTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGG
1- Different target sequences with PAM-in and PAM-out orientations.
2- Possible combinations of pairs of AAVS1 gRNAs in PAM-in and PAM-out orientations
and spacer distances:
36
Human gRNA under the U6 promoter:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAAT
TAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAA
TTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAA
CTTGAAAGTATTTCGATTTCTTGGGTTTATATATCTTGTGGAAAGGACgNNNNNNNNNNNNNN
NNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA
AGTGGCACCGAGTCGGTGCTTTTTTT
(U6 promoter sequence is highlighted in red; guide sequence is highlighted in green, the gR
NA scaffold in purple, and the terminator fragment is highlighted in black)
37
Construct DNA and amino acid sequences used in this study.
1- dCas9
1-1-
DNA sequence:
ATGGACAAGAAGTATTCTATCGGACTGGCCATCGGGACTAATAGCGTCGGGTGGGCCGTGATCAC
TGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTGCTCGGGAACACCGACCGGCATTCCATCA
AGAAAAATCTGATCGGAGCTCTCCTCTTTGATTCAGGGGAGACCGCTGAAGCAACCCGCCTCAAG
CGGACTGCTAGACGGCGGTACACCAGGAGGAAGAACCGGATTTGTTACCTTCAAGAGATATTCTC
CAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCATAGGCTGGAAGAATCATTCCTCGTGGAAG
AGGATAAGAAGCATGAACGGCATCCCATCTTCGGTAATATCGTCGACGAGGTGGCCTATCACGAG
AAATACCCAACCATCTACCATCTTCGCAAAAAGCTGGTGGACTCAACCGACAAGGCAGACCTCCG
GCTTATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCGACC
TCAATCCTGACAATAGCGATGTGGATAAACTGTTCATCCAGCTGGTGCAGACTTACAACCAGCTC
TTTGAAGAGAACCCCATCAATGCAAGCGGAGTCGATGCCAAGGCCATTCTGTCAGCCCGGCTGTC
AAAGAGCCGCAGACTTGAGAATCTTATCGCTCAGCTGCCGGGTGAAAAGAAAAATGGACTGTTC
GGGAACCTGATTGCTCTTTCACTTGGGCTGACTCCCAATTTCAAGTCTAATTTCGACCTGGCAGA
GGATGCCAAGCTGCAACTGTCCAAGGACACCTATGATGACGATCTCGACAACCTCCTGGCCCAGA
TCGGTGACCAATACGCCGACCTTTTCCTTGCTGCTAAGAATCTTTCTGACGCCATCCTGCTGTCT
GACATTCTCCGCGTGAACACTGAAATCACCAAGGCCCCTCTTTCAGCTTCAATGATTAAGCGGTA
TGATGAGCACCACCAGGACCTGACCCTGCTTAAGGCACTCGTCCGGCAGCAGCTTCCGGAGAAGT
ACAAGGAAATCTTCTTTGACCAGTCAAAGAATGGATACGCCGGCTACATCGACGGAGGTGCCTCC
CAAGAGGAATTTTATAAGTTTATCAAACCTATCCTTGAGAAGATGGACGGCACCGAAGAGCTCC
TCGTGAAACTGAATCGGGAGGATCTGCTGCGGAAGCAGCGCACTTTCGACAATGGGAGCATTCCC
CACCAGATCCATCTTGGGGAGCTTCACGCCATCCTTCGGCGCCAAGAGGACTTCTACCCCTTTCTT
AAGGACAACAGGGAGAAGATTGAGAAAATTCTCACTTTCCGCATCCCCTACTACGTGGGACCCCT
CGCCAGAGGAAATAGCCGGTTTGCTTGGATGACCAGAAAGTCAGAAGAAACTATCACTCCCTGG
AACTTCGAAGAGGTGGTGGACAAGGGAGCCAGCGCTCAGTCATTCATCGAACGGATGACTAACT
TCGATAAGAACCTCCCCAATGAGAAGGTCCTGCCGAAACATTCCCTGCTCTACGAGTACTTTACC
GTGTACAACGAGCTGACCAAGGTGAAATATGTCACCGAAGGGATGAGGAAGCCCGCATTCCTGTC
AGGCGAACAAAAGAAGGCAATTGTGGACCTTCTGTTCAAGACCAATAGAAAGGTGACCGTGAAG
CAGCTGAAGGAGGACTATTTCAAGAAAATTGAATGCTTCGACTCTGTGGAGATTAGCGGGGTCG
38
AAGATCGGTTCAACGCAAGCCTGGGTACCTACCATGATCTGCTTAAGATCATCAAGGACAAGGAT
TTTCTGGACAATGAGGAGAACGAGGACATCCTTGAGGACATTGTCCTGACTCTCACTCTGTTCGA
GGACCGGGAAATGATCGAGGAGAGGCTTAAGACCTACGCCCATCTGTTCGACGATAAAGTGATG
AAGCAACTTAAACGGAGAAGATATACCGGATGGGGACGCCTTAGCCGCAAACTCATCAACGGAA
TCCGGGACAAACAGAGCGGAAAGACCATTCTTGATTTCCTTAAGAGCGACGGATTCGCTAATCGC
AACTTCATGCAACTTATCCATGATGATTCCCTGACCTTTAAGGAGGACATCCAGAAGGCCCAAGT
GTCTGGACAAGGTGACTCACTGCACGAGCATATCGCAAATCTGGCTGGTTCACCCGCTATTAAGA
AGGGTATTCTCCAGACCGTGAAAGTCGTGGACGAGCTGGTCAAGGTGATGGGTCGCCATAAACC
AGAGAACATTGTCATCGAGATGGCCAGGGAAAACCAGACTACCCAGAAGGGACAGAAGAACAGC
AGGGAGCGGATGAAAAGAATTGAGGAAGGGATTAAGGAGCTCGGGTCACAGATCCTTAAAGAGC
ACCCGGTGGAAAACACCCAGCTTCAGAATGAGAAGCTCTATCTGTACTACCTTCAAAATGGACGC
GATATGTATGTGGACCAAGAGCTTGATATCAACAGGCTCTCAGACTACGACGTGGACGCCATCGT
CCCTCAGAGCTTCCTCAAAGACGACTCAATTGACAATAAGGTGCTGACTCGCTCAGACAAGAACC
GGGGAAAGTCAGATAACGTGCCCTCAGAGGAAGTCGTGAAAAAGATGAAGAACTATTGGCGCCA
GCTTCTGAACGCAAAGCTGATCACTCAGCGGAAGTTCGACAATCTCACTAAGGCTGAGAGGGGCG
GACTGAGCGAACTGGACAAAGCAGGATTCATTAAACGGCAACTTGTGGAGACTCGGCAGATTAC
TAAACATGTCGCCCAAATCCTTGACTCACGCATGAATACCAAGTACGACGAAAACGACAAACTTA
TCCGCGAGGTGAAGGTGATTACCCTGAAGTCCAAGCTGGTCAGCGATTTCAGAAAGGACTTTCAA
TTCTACAAAGTGCGGGAGATCAATAACTATCATCATGCTCATGACGCATATCTGAATGCCGTGGT
GGGAACCGCCCTGATCAAGAAGTACCCAAAGCTGGAAAGCGAGTTCGTGTACGGAGACTACAAG
GTCTACGACGTGCGCAAGATGATTGCCAAATCTGAGCAGGAGATCGGAAAGGCCACCGCAAAGT
ACTTCTTCTACAGCAACATCATGAATTTCTTCAAGACCGAAATCACCCTTGCAAACGGTGAGATC
CGGAAGAGGCCGCTCATCGAGACTAATGGGGAGACTGGCGAAATCGTGTGGGACAAGGGCAGAG
ATTTCGCTACCGTGCGCAAAGTGCTTTCTATGCCTCAAGTGAACATCGTGAAGAAAACCGAGGTG
CAAACCGGAGGCTTTTCTAAGGAATCAATCCTCCCCAAGCGCAACTCCGACAAGCTCATTGCAAG
GAAGAAGGATTGGGACCCTAAGAAGTACGGCGGATTCGATTCACCAACTGTGGCTTATTCTGTCC
TGGTCGTGGCTAAGGTGGAAAAAGGAAAGTCTAAGAAGCTCAAGAGCGTGAAGGAACTGCTGGG
TATCACCATTATGGAGCGCAGCTCCTTCGAGAAGAACCCAATTGACTTTCTCGAAGCCAAAGGTT
ACAAGGAAGTCAAGAAGGACCTTATCATCAAGCTCCCAAAGTATAGCCTGTTCGAACTGGAGAA
TGGGCGGAAGCGGATGCTCGCCTCCGCTGGCGAACTTCAGAAGGGTAATGAGCTGGCTCTCCCCT
CCAAGTACGTGAATTTCCTCTACCTTGCAAGCCATTACGAGAAGCTGAAGGGGAGCCCCGAGGAC
AACGAGCAAAAGCAACTGTTTGTGGAGCAGCATAAGCATTATCTGGACGAGATCATTGAGCAGA
TTTCCGAGTTTTCTAAACGCGTCATTCTCGCTGATGCCAACCTCGATAAAGTCCTTAGCGCATAC
AATAAGCACAGAGACAAACCAATTCGGGAGCAGGCTGAGAATATCATCCACCTGTTCACCCTCAC
CAATCTTGGTGCCCCTGCCGCATTCAAGTACTTCGACACCACCATCGACCGGAAACGCTATACCT
CCACCAAAGAAGTGCTGGACGCCACCCTCATCCACCAGAGCATCACCGGACTTTACGAAACTCGG
ATTGACCTCTCACAGCTCGGAGGGGATGAGGGAGCTGATCCAAAAAAGAAGAGAAAGGTAGATC
CAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTATAG
2-239
dCas9 amino acid sequence:
MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL
KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVA
YHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQT
YNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF
DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS
MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP
YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK
HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE
CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD
SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA
RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ
ELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLN
AKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR
EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK
VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR
DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY
SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELE
NGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEII
EQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK
RYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDEGADPKKKRKVDPKKKRKVDPKKKRKV
3- fdCas9 DNA and amino acid sequence
1-2- DNA sequence
ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATG
ACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTGGAGGTTCTAT
GGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAA
TATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTC
TTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGG
ATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTG
GATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGAT
ATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCC
ATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCT
CAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTT
TAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTCAGACGGAAATTTAA
TAACGGCGAGATAAACTTTAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACCCGAAA
CCATGGACAAGAAGTATTCTATCGGACTGGCCATCGGGACTAATAGCGTCGGGTGGGCCGTGATC
ACTGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTGCTCGGGAACACCGACCGGCATTCCAT
40
CAAGAAAAATCTGATCGGAGCTCTCCTCTTTGATTCAGGGGAGACCGCTGAAGCAACCCGCCTCA
AGCGGACTGCTAGACGGCGGTACACCAGGAGGAAGAACCGGATTTGTTACCTTCAAGAGATATT
CTCCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCATAGGCTGGAAGAATCATTCCTCGTGG
AAGAGGATAAGAAGCATGAACGGCATCCCATCTTCGGTAATATCGTCGACGAGGTGGCCTATCAC
GAGAAATACCCAACCATCTACCATCTTCGCAAAAAGCTGGTGGACTCAACCGACAAGGCAGACCT
CCGGCTTATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCG
ACCTCAATCCTGACAATAGCGATGTGGATAAACTGTTCATCCAGCTGGTGCAGACTTACAACCAG
CTCTTTGAAGAGAACCCCATCAATGCAAGCGGAGTCGATGCCAAGGCCATTCTGTCAGCCCGGCT
GTCAAAGAGCCGCAGACTTGAGAATCTTATCGCTCAGCTGCCGGGTGAAAAGAAAAATGGACTG
TTCGGGAACCTGATTGCTCTTTCACTTGGGCTGACTCCCAATTTCAAGTCTAATTTCGACCTGGC
AGAGGATGCCAAGCTGCAACTGTCCAAGGACACCTATGATGACGATCTCGACAACCTCCTGGCCC
AGATCGGTGACCAATACGCCGACCTTTTCCTTGCTGCTAAGAATCTTTCTGACGCCATCCTGCTG
TCTGACATTCTCCGCGTGAACACTGAAATCACCAAGGCCCCTCTTTCAGCTTCAATGATTAAGCG
GTATGATGAGCACCACCAGGACCTGACCCTGCTTAAGGCACTCGTCCGGCAGCAGCTTCCGGAGA
AGTACAAGGAAATCTTCTTTGACCAGTCAAAGAATGGATACGCCGGCTACATCGACGGAGGTGCC
TCCCAAGAGGAATTTTATAAGTTTATCAAACCTATCCTTGAGAAGATGGACGGCACCGAAGAGC
TCCTCGTGAAACTGAATCGGGAGGATCTGCTGCGGAAGCAGCGCACTTTCGACAATGGGAGCATT
CCCCACCAGATCCATCTTGGGGAGCTTCACGCCATCCTTCGGCGCCAAGAGGACTTCTACCCCTTT
CTTAAGGACAACAGGGAGAAGATTGAGAAAATTCTCACTTTCCGCATCCCCTACTACGTGGGACC
CCTCGCCAGAGGAAATAGCCGGTTTGCTTGGATGACCAGAAAGTCAGAAGAAACTATCACTCCCT
GGAACTTCGAAGAGGTGGTGGACAAGGGAGCCAGCGCTCAGTCATTCATCGAACGGATGACTAA
CTTCGATAAGAACCTCCCCAATGAGAAGGTCCTGCCGAAACATTCCCTGCTCTACGAGTACTTTA
CCGTGTACAACGAGCTGACCAAGGTGAAATATGTCACCGAAGGGATGAGGAAGCCCGCATTCCTG
TCAGGCGAACAAAAGAAGGCAATTGTGGACCTTCTGTTCAAGACCAATAGAAAGGTGACCGTGA
AGCAGCTGAAGGAGGACTATTTCAAGAAAATTGAATGCTTCGACTCTGTGGAGATTAGCGGGGT
CGAAGATCGGTTCAACGCAAGCCTGGGTACCTACCATGATCTGCTTAAGATCATCAAGGACAAGG
ATTTTCTGGACAATGAGGAGAACGAGGACATCCTTGAGGACATTGTCCTGACTCTCACTCTGTTC
GAGGACCGGGAAATGATCGAGGAGAGGCTTAAGACCTACGCCCATCTGTTCGACGATAAAGTGA
TGAAGCAACTTAAACGGAGAAGATATACCGGATGGGGACGCCTTAGCCGCAAACTCATCAACGG
AATCCGGGACAAACAGAGCGGAAAGACCATTCTTGATTTCCTTAAGAGCGACGGATTCGCTAATC
GCAACTTCATGCAACTTATCCATGATGATTCCCTGACCTTTAAGGAGGACATCCAGAAGGCCCAA
GTGTCTGGACAAGGTGACTCACTGCACGAGCATATCGCAAATCTGGCTGGTTCACCCGCTATTAA
GAAGGGTATTCTCCAGACCGTGAAAGTCGTGGACGAGCTGGTCAAGGTGATGGGTCGCCATAAA
CCAGAGAACATTGTCATCGAGATGGCCAGGGAAAACCAGACTACCCAGAAGGGACAGAAGAACA
GCAGGGAGCGGATGAAAAGAATTGAGGAAGGGATTAAGGAGCTCGGGTCACAGATCCTTAAAGA
GCACCCGGTGGAAAACACCCAGCTTCAGAATGAGAAGCTCTATCTGTACTACCTTCAAAATGGAC
GCGATATGTATGTGGACCAAGAGCTTGATATCAACAGGCTCTCAGACTACGACGTGGACGCCATC
GTCCCTCAGAGCTTCCTCAAAGACGACTCAATTGACAATAAGGTGCTGACTCGCTCAGACAAGAA
CCGGGGAAAGTCAGATAACGTGCCCTCAGAGGAAGTCGTGAAAAAGATGAAGAACTATTGGCGC
CAGCTTCTGAACGCAAAGCTGATCACTCAGCGGAAGTTCGACAATCTCACTAAGGCTGAGAGGGG
41
CGGACTGAGCGAACTGGACAAAGCAGGATTCATTAAACGGCAACTTGTGGAGACTCGGCAGATT
ACTAAACATGTCGCCCAAATCCTTGACTCACGCATGAATACCAAGTACGACGAAAACGACAAACT
TATCCGCGAGGTGAAGGTGATTACCCTGAAGTCCAAGCTGGTCAGCGATTTCAGAAAGGACTTTC
AATTCTACAAAGTGCGGGAGATCAATAACTATCATCATGCTCATGACGCATATCTGAATGCCGTG
GTGGGAACCGCCCTGATCAAGAAGTACCCAAAGCTGGAAAGCGAGTTCGTGTACGGAGACTACA
AGGTCTACGACGTGCGCAAGATGATTGCCAAATCTGAGCAGGAGATCGGAAAGGCCACCGCAAA
GTACTTCTTCTACAGCAACATCATGAATTTCTTCAAGACCGAAATCACCCTTGCAAACGGTGAGA
TCCGGAAGAGGCCGCTCATCGAGACTAATGGGGAGACTGGCGAAATCGTGTGGGACAAGGGCAG
AGATTTCGCTACCGTGCGCAAAGTGCTTTCTATGCCTCAAGTGAACATCGTGAAGAAAACCGAGG
TGCAAACCGGAGGCTTTTCTAAGGAATCAATCCTCCCCAAGCGCAACTCCGACAAGCTCATTGCA
AGGAAGAAGGATTGGGACCCTAAGAAGTACGGCGGATTCGATTCACCAACTGTGGCTTATTCTG
TCCTGGTCGTGGCTAAGGTGGAAAAAGGAAAGTCTAAGAAGCTCAAGAGCGTGAAGGAACTGCT
GGGTATCACCATTATGGAGCGCAGCTCCTTCGAGAAGAACCCAATTGACTTTCTCGAAGCCAAAG
GTTACAAGGAAGTCAAGAAGGACCTTATCATCAAGCTCCCAAAGTATAGCCTGTTCGAACTGGA
GAATGGGCGGAAGCGGATGCTCGCCTCCGCTGGCGAACTTCAGAAGGGTAATGAGCTGGCTCTCC
CCTCCAAGTACGTGAATTTCCTCTACCTTGCAAGCCATTACGAGAAGCTGAAGGGGAGCCCCGAG
GACAACGAGCAAAAGCAACTGTTTGTGGAGCAGCATAAGCATTATCTGGACGAGATCATTGAGC
AGATTTCCGAGTTTTCTAAACGCGTCATTCTCGCTGATGCCAACCTCGATAAAGTCCTTAGCGCA
TACAATAAGCACAGAGACAAACCAATTCGGGAGCAGGCTGAGAATATCATCCACCTGTTCACCCT
CACCAATCTTGGTGCCCCTGCCGCATTCAAGTACTTCGACACCACCATCGACCGGAAACGCTATA
CCTCCACCAAAGAAGTGCTGGACGCCACCCTCATCCACCAGAGCATCACCGGACTTTACGAAACT
CGGATTGACCTCTCACAGCTCGGAGGGGATGAGGGAGCTGATCCAAAAAAGAAGAGAAAGGTAG
ATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTATAG
1-3-
Amino acid sequence
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPGGSMGSQLVKSELEEKKSELRHKL
KYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDT
KAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQL
TRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFSGSETPGTSESATPETMDK
KYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR
RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY
HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN
ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD
TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL
KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK
QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR
KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE
GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS
42
RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS
DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVET
RQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG
EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA
RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY
KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNE
QKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDEGADPKKKRKVDPKKKRK
VDPKKKRKV*
2- Sharky.fdCas9
2-2-
DNA sequence
ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATG
ACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTGGAGGTTCTAT
GGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAA
TATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATCCCACTCAGGATAGAATTC
TTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTGAACATTTGGGTGG
ATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTG
GATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGAT
ATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCC
ATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCT
CAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTT
TAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAA
TAACGGCGAGATAAACTTTAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACCCGAAA
CCATGGACAAGAAGTATTCTATCGGACTGGCCATCGGGACTAATAGCGTCGGGTGGGCCGTGATC
ACTGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTGCTCGGGAACACCGACCGGCATTCCAT
CAAGAAAAATCTGATCGGAGCTCTCCTCTTTGATTCAGGGGAGACCGCTGAAGCAACCCGCCTCA
AGCGGACTGCTAGACGGCGGTACACCAGGAGGAAGAACCGGATTTGTTACCTTCAAGAGATATT
CTCCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCATAGGCTGGAAGAATCATTCCTCGTGG
AAGAGGATAAGAAGCATGAACGGCATCCCATCTTCGGTAATATCGTCGACGAGGTGGCCTATCAC
GAGAAATACCCAACCATCTACCATCTTCGCAAAAAGCTGGTGGACTCAACCGACAAGGCAGACCT
CCGGCTTATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCG
ACCTCAATCCTGACAATAGCGATGTGGATAAACTGTTCATCCAGCTGGTGCAGACTTACAACCAG
CTCTTTGAAGAGAACCCCATCAATGCAAGCGGAGTCGATGCCAAGGCCATTCTGTCAGCCCGGCT
GTCAAAGAGCCGCAGACTTGAGAATCTTATCGCTCAGCTGCCGGGTGAAAAGAAAAATGGACTG
TTCGGGAACCTGATTGCTCTTTCACTTGGGCTGACTCCCAATTTCAAGTCTAATTTCGACCTGGC
43
AGAGGATGCCAAGCTGCAACTGTCCAAGGACACCTATGATGACGATCTCGACAACCTCCTGGCCC
AGATCGGTGACCAATACGCCGACCTTTTCCTTGCTGCTAAGAATCTTTCTGACGCCATCCTGCTG
TCTGACATTCTCCGCGTGAACACTGAAATCACCAAGGCCCCTCTTTCAGCTTCAATGATTAAGCG
GTATGATGAGCACCACCAGGACCTGACCCTGCTTAAGGCACTCGTCCGGCAGCAGCTTCCGGAGA
AGTACAAGGAAATCTTCTTTGACCAGTCAAAGAATGGATACGCCGGCTACATCGACGGAGGTGCC
TCCCAAGAGGAATTTTATAAGTTTATCAAACCTATCCTTGAGAAGATGGACGGCACCGAAGAGC
TCCTCGTGAAACTGAATCGGGAGGATCTGCTGCGGAAGCAGCGCACTTTCGACAATGGGAGCATT
CCCCACCAGATCCATCTTGGGGAGCTTCACGCCATCCTTCGGCGCCAAGAGGACTTCTACCCCTTT
CTTAAGGACAACAGGGAGAAGATTGAGAAAATTCTCACTTTCCGCATCCCCTACTACGTGGGACC
CCTCGCCAGAGGAAATAGCCGGTTTGCTTGGATGACCAGAAAGTCAGAAGAAACTATCACTCCCT
GGAACTTCGAAGAGGTGGTGGACAAGGGAGCCAGCGCTCAGTCATTCATCGAACGGATGACTAA
CTTCGATAAGAACCTCCCCAATGAGAAGGTCCTGCCGAAACATTCCCTGCTCTACGAGTACTTTA
CCGTGTACAACGAGCTGACCAAGGTGAAATATGTCACCGAAGGGATGAGGAAGCCCGCATTCCTG
TCAGGCGAACAAAAGAAGGCAATTGTGGACCTTCTGTTCAAGACCAATAGAAAGGTGACCGTGA
AGCAGCTGAAGGAGGACTATTTCAAGAAAATTGAATGCTTCGACTCTGTGGAGATTAGCGGGGT
CGAAGATCGGTTCAACGCAAGCCTGGGTACCTACCATGATCTGCTTAAGATCATCAAGGACAAGG
ATTTTCTGGACAATGAGGAGAACGAGGACATCCTTGAGGACATTGTCCTGACTCTCACTCTGTTC
GAGGACCGGGAAATGATCGAGGAGAGGCTTAAGACCTACGCCCATCTGTTCGACGATAAAGTGA
TGAAGCAACTTAAACGGAGAAGATATACCGGATGGGGACGCCTTAGCCGCAAACTCATCAACGG
AATCCGGGACAAACAGAGCGGAAAGACCATTCTTGATTTCCTTAAGAGCGACGGATTCGCTAATC
GCAACTTCATGCAACTTATCCATGATGATTCCCTGACCTTTAAGGAGGACATCCAGAAGGCCCAA
GTGTCTGGACAAGGTGACTCACTGCACGAGCATATCGCAAATCTGGCTGGTTCACCCGCTATTAA
GAAGGGTATTCTCCAGACCGTGAAAGTCGTGGACGAGCTGGTCAAGGTGATGGGTCGCCATAAA
CCAGAGAACATTGTCATCGAGATGGCCAGGGAAAACCAGACTACCCAGAAGGGACAGAAGAACA
GCAGGGAGCGGATGAAAAGAATTGAGGAAGGGATTAAGGAGCTCGGGTCACAGATCCTTAAAGA
GCACCCGGTGGAAAACACCCAGCTTCAGAATGAGAAGCTCTATCTGTACTACCTTCAAAATGGAC
GCGATATGTATGTGGACCAAGAGCTTGATATCAACAGGCTCTCAGACTACGACGTGGACGCCATC
GTCCCTCAGAGCTTCCTCAAAGACGACTCAATTGACAATAAGGTGCTGACTCGCTCAGACAAGAA
CCGGGGAAAGTCAGATAACGTGCCCTCAGAGGAAGTCGTGAAAAAGATGAAGAACTATTGGCGC
CAGCTTCTGAACGCAAAGCTGATCACTCAGCGGAAGTTCGACAATCTCACTAAGGCTGAGAGGGG
CGGACTGAGCGAACTGGACAAAGCAGGATTCATTAAACGGCAACTTGTGGAGACTCGGCAGATT
ACTAAACATGTCGCCCAAATCCTTGACTCACGCATGAATACCAAGTACGACGAAAACGACAAACT
TATCCGCGAGGTGAAGGTGATTACCCTGAAGTCCAAGCTGGTCAGCGATTTCAGAAAGGACTTTC
AATTCTACAAAGTGCGGGAGATCAATAACTATCATCATGCTCATGACGCATATCTGAATGCCGTG
GTGGGAACCGCCCTGATCAAGAAGTACCCAAAGCTGGAAAGCGAGTTCGTGTACGGAGACTACA
AGGTCTACGACGTGCGCAAGATGATTGCCAAATCTGAGCAGGAGATCGGAAAGGCCACCGCAAA
GTACTTCTTCTACAGCAACATCATGAATTTCTTCAAGACCGAAATCACCCTTGCAAACGGTGAGA
TCCGGAAGAGGCCGCTCATCGAGACTAATGGGGAGACTGGCGAAATCGTGTGGGACAAGGGCAG
AGATTTCGCTACCGTGCGCAAAGTGCTTTCTATGCCTCAAGTGAACATCGTGAAGAAAACCGAGG
TGCAAACCGGAGGCTTTTCTAAGGAATCAATCCTCCCCAAGCGCAACTCCGACAAGCTCATTGCA
44
AGGAAGAAGGATTGGGACCCTAAGAAGTACGGCGGATTCGATTCACCAACTGTGGCTTATTCTG
TCCTGGTCGTGGCTAAGGTGGAAAAAGGAAAGTCTAAGAAGCTCAAGAGCGTGAAGGAACTGCT
GGGTATCACCATTATGGAGCGCAGCTCCTTCGAGAAGAACCCAATTGACTTTCTCGAAGCCAAAG
GTTACAAGGAAGTCAAGAAGGACCTTATCATCAAGCTCCCAAAGTATAGCCTGTTCGAACTGGA
GAATGGGCGGAAGCGGATGCTCGCCTCCGCTGGCGAACTTCAGAAGGGTAATGAGCTGGCTCTCC
CCTCCAAGTACGTGAATTTCCTCTACCTTGCAAGCCATTACGAGAAGCTGAAGGGGAGCCCCGAG
GACAACGAGCAAAAGCAACTGTTTGTGGAGCAGCATAAGCATTATCTGGACGAGATCATTGAGC
AGATTTCCGAGTTTTCTAAACGCGTCATTCTCGCTGATGCCAACCTCGATAAAGTCCTTAGCGCA
TACAATAAGCACAGAGACAAACCAATTCGGGAGCAGGCTGAGAATATCATCCACCTGTTCACCCT
CACCAATCTTGGTGCCCCTGCCGCATTCAAGTACTTCGACACCACCATCGACCGGAAACGCTATA
CCTCCACCAAAGAAGTGCTGGACGCCACCCTCATCCACCAGAGCATCACCGGACTTTACGAAACT
CGGATTGACCTCTCACAGCTCGGAGGGGATGAGGGAGCTGATCCAAAAAAGAAGAGAAAGGTAG
ATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTATAG
2-3-
Amino acid sequence
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPGGSMGSQLVKSELEEKKSELRHKL
KYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDT
KAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQL
TRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFSGSETPGTSESATPETMDK
KYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR
RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY
HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN
ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD
TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL
KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK
QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR
KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE
GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS
RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS
DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVET
RQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG
EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA
RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY
KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNE
45
QKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDEGADPKKKRKVDPKKKRK
VDPKKKRKV*
3- dCas9f (3GS-3NLS)
3-2- DNA sequence
ATGGACAAGAAGTATTCTATCGGACTGGCCATCGGGACTAATAGCGTCGGGTGGGCCGTGATCAC
TGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTGCTCGGGAACACCGACCGGCATTCCATCA
AGAAAAATCTGATCGGAGCTCTCCTCTTTGATTCAGGGGAGACCGCTGAAGCAACCCGCCTCAAG
CGGACTGCTAGACGGCGGTACACCAGGAGGAAGAACCGGATTTGTTACCTTCAAGAGATATTCTC
CAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCATAGGCTGGAAGAATCATTCCTCGTGGAAG
AGGATAAGAAGCATGAACGGCATCCCATCTTCGGTAATATCGTCGACGAGGTGGCCTATCACGAG
AAATACCCAACCATCTACCATCTTCGCAAAAAGCTGGTGGACTCAACCGACAAGGCAGACCTCCG
GCTTATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCGACC
TCAATCCTGACAATAGCGATGTGGATAAACTGTTCATCCAGCTGGTGCAGACTTACAACCAGCTC
TTTGAAGAGAACCCCATCAATGCAAGCGGAGTCGATGCCAAGGCCATTCTGTCAGCCCGGCTGTC
AAAGAGCCGCAGACTTGAGAATCTTATCGCTCAGCTGCCGGGTGAAAAGAAAAATGGACTGTTC
GGGAACCTGATTGCTCTTTCACTTGGGCTGACTCCCAATTTCAAGTCTAATTTCGACCTGGCAGA
GGATGCCAAGCTGCAACTGTCCAAGGACACCTATGATGACGATCTCGACAACCTCCTGGCCCAGA
TCGGTGACCAATACGCCGACCTTTTCCTTGCTGCTAAGAATCTTTCTGACGCCATCCTGCTGTCT
GACATTCTCCGCGTGAACACTGAAATCACCAAGGCCCCTCTTTCAGCTTCAATGATTAAGCGGTA
TGATGAGCACCACCAGGACCTGACCCTGCTTAAGGCACTCGTCCGGCAGCAGCTTCCGGAGAAGT
ACAAGGAAATCTTCTTTGACCAGTCAAAGAATGGATACGCCGGCTACATCGACGGAGGTGCCTCC
CAAGAGGAATTTTATAAGTTTATCAAACCTATCCTTGAGAAGATGGACGGCACCGAAGAGCTCC
TCGTGAAACTGAATCGGGAGGATCTGCTGCGGAAGCAGCGCACTTTCGACAATGGGAGCATTCCC
CACCAGATCCATCTTGGGGAGCTTCACGCCATCCTTCGGCGCCAAGAGGACTTCTACCCCTTTCTT
AAGGACAACAGGGAGAAGATTGAGAAAATTCTCACTTTCCGCATCCCCTACTACGTGGGACCCCT
CGCCAGAGGAAATAGCCGGTTTGCTTGGATGACCAGAAAGTCAGAAGAAACTATCACTCCCTGG
AACTTCGAAGAGGTGGTGGACAAGGGAGCCAGCGCTCAGTCATTCATCGAACGGATGACTAACT
TCGATAAGAACCTCCCCAATGAGAAGGTCCTGCCGAAACATTCCCTGCTCTACGAGTACTTTACC
GTGTACAACGAGCTGACCAAGGTGAAATATGTCACCGAAGGGATGAGGAAGCCCGCATTCCTGTC
AGGCGAACAAAAGAAGGCAATTGTGGACCTTCTGTTCAAGACCAATAGAAAGGTGACCGTGAAG
CAGCTGAAGGAGGACTATTTCAAGAAAATTGAATGCTTCGACTCTGTGGAGATTAGCGGGGTCG
AAGATCGGTTCAACGCAAGCCTGGGTACCTACCATGATCTGCTTAAGATCATCAAGGACAAGGAT
TTTCTGGACAATGAGGAGAACGAGGACATCCTTGAGGACATTGTCCTGACTCTCACTCTGTTCGA
GGACCGGGAAATGATCGAGGAGAGGCTTAAGACCTACGCCCATCTGTTCGACGATAAAGTGATG
AAGCAACTTAAACGGAGAAGATATACCGGATGGGGACGCCTTAGCCGCAAACTCATCAACGGAA
TCCGGGACAAACAGAGCGGAAAGACCATTCTTGATTTCCTTAAGAGCGACGGATTCGCTAATCGC
AACTTCATGCAACTTATCCATGATGATTCCCTGACCTTTAAGGAGGACATCCAGAAGGCCCAAGT
46
GTCTGGACAAGGTGACTCACTGCACGAGCATATCGCAAATCTGGCTGGTTCACCCGCTATTAAGA
AGGGTATTCTCCAGACCGTGAAAGTCGTGGACGAGCTGGTCAAGGTGATGGGTCGCCATAAACC
AGAGAACATTGTCATCGAGATGGCCAGGGAAAACCAGACTACCCAGAAGGGACAGAAGAACAGC
AGGGAGCGGATGAAAAGAATTGAGGAAGGGATTAAGGAGCTCGGGTCACAGATCCTTAAAGAGC
ACCCGGTGGAAAACACCCAGCTTCAGAATGAGAAGCTCTATCTGTACTACCTTCAAAATGGACGC
GATATGTATGTGGACCAAGAGCTTGATATCAACAGGCTCTCAGACTACGACGTGGACGCCATCGT
CCCTCAGAGCTTCCTCAAAGACGACTCAATTGACAATAAGGTGCTGACTCGCTCAGACAAGAACC
GGGGAAAGTCAGATAACGTGCCCTCAGAGGAAGTCGTGAAAAAGATGAAGAACTATTGGCGCCA
GCTTCTGAACGCAAAGCTGATCACTCAGCGGAAGTTCGACAATCTCACTAAGGCTGAGAGGGGCG
GACTGAGCGAACTGGACAAAGCAGGATTCATTAAACGGCAACTTGTGGAGACTCGGCAGATTAC
TAAACATGTCGCCCAAATCCTTGACTCACGCATGAATACCAAGTACGACGAAAACGACAAACTTA
TCCGCGAGGTGAAGGTGATTACCCTGAAGTCCAAGCTGGTCAGCGATTTCAGAAAGGACTTTCAA
TTCTACAAAGTGCGGGAGATCAATAACTATCATCATGCTCATGACGCATATCTGAATGCCGTGGT
GGGAACCGCCCTGATCAAGAAGTACCCAAAGCTGGAAAGCGAGTTCGTGTACGGAGACTACAAG
GTCTACGACGTGCGCAAGATGATTGCCAAATCTGAGCAGGAGATCGGAAAGGCCACCGCAAAGT
ACTTCTTCTACAGCAACATCATGAATTTCTTCAAGACCGAAATCACCCTTGCAAACGGTGAGATC
CGGAAGAGGCCGCTCATCGAGACTAATGGGGAGACTGGCGAAATCGTGTGGGACAAGGGCAGAG
ATTTCGCTACCGTGCGCAAAGTGCTTTCTATGCCTCAAGTGAACATCGTGAAGAAAACCGAGGTG
CAAACCGGAGGCTTTTCTAAGGAATCAATCCTCCCCAAGCGCAACTCCGACAAGCTCATTGCAAG
GAAGAAGGATTGGGACCCTAAGAAGTACGGCGGATTCGATTCACCAACTGTGGCTTATTCTGTCC
TGGTCGTGGCTAAGGTGGAAAAAGGAAAGTCTAAGAAGCTCAAGAGCGTGAAGGAACTGCTGGG
TATCACCATTATGGAGCGCAGCTCCTTCGAGAAGAACCCAATTGACTTTCTCGAAGCCAAAGGTT
ACAAGGAAGTCAAGAAGGACCTTATCATCAAGCTCCCAAAGTATAGCCTGTTCGAACTGGAGAA
TGGGCGGAAGCGGATGCTCGCCTCCGCTGGCGAACTTCAGAAGGGTAATGAGCTGGCTCTCCCCT
CCAAGTACGTGAATTTCCTCTACCTTGCAAGCCATTACGAGAAGCTGAAGGGGAGCCCCGAGGAC
AACGAGCAAAAGCAACTGTTTGTGGAGCAGCATAAGCATTATCTGGACGAGATCATTGAGCAGA
TTTCCGAGTTTTCTAAACGCGTCATTCTCGCTGATGCCAACCTCGATAAAGTCCTTAGCGCATAC
AATAAGCACAGAGACAAACCAATTCGGGAGCAGGCTGAGAATATCATCCACCTGTTCACCCTCAC
CAATCTTGGTGCCCCTGCCGCATTCAAGTACTTCGACACCACCATCGACCGGAAACGCTATACCT
CCACCAAAGAAGTGCTGGACGCCACCCTCATCCACCAGAGCATCACCGGACTTTACGAAACTCGG
ATTGACCTCTCACAGCTCGGAGGGGATGAGGGAGCTGATGATCCAAAAAAGAAGAGAAAGGTAG
ATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGGGCCcGGTTCCGGTTC
CGGTTCCCAACTCGTGAAGAGTGAACTTGAGGAGAAAAAGTCGGAGCTGCGGCACAAATTGAAA
TACGTACCGCATGAATACATCGAACTTATCGAAATTGCTAGGAACTCGACTCAAGACAGAATCCT
TGAGATGAAGGTAATGGAGTTCTTTATGAAGGTTTATGGATACCGAGGGAAGCATCTCGGTGGA
TCACGAAAACCCGACGGAGCAATCTATACGGTGGGGAGCCCGATTGATTACGGAGTGATCGTCGA
CACGAAAGCCTACAGCGGTGGGTACAATCTTCCCATCGGGCAGGCAGATGAGATGCAACGTTATG
TCGAAGAAAATCAGACCAGGAACAAACACATCAATCCAAATGAGTGGTGGAAAGTGTATCCTTC
ATCAGTGACCGAGTTTAAGTTTTTGTTTGTCTCTGGGCATTTCAAAGGCAACTATAAGGCCCAGC
TCACACGGTTGAATCACATTACGAACTGCAATGGTGCGGTTTTGTCCGTAGAGGAACTGCTCATT
47
GGTGGAGAAATGATCAAAGCGGGAACTCTGACACTGGAAGAAGTCAGACGCAAGTTTAACAATG
GCGAGATCAATTTCCGCTCATAA
48
3-3-
Amino acid sequence
MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY
PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ
LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD
LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDL
LRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW
MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY
VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH
DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG
RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL
AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS
QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLT
RSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV
ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA
YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN
GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI
ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG
YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN
EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG
APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDEGADDPKKKRKVDPKKK
RKVDPKKKRKVGPGSGSGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVME
FFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTR
NKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKA
GTLTLEEVRRKFNNGEINFRS*
4- dCas9f (3GS-3NLS)
4-2- DNA sequence
ATGGACAAGAAGTATTCTATCGGACTGGCCATCGGGACTAATAGCGTCGGGTGGGCCGTGATCAC
TGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTGCTCGGGAACACCGACCGGCATTCCATCA
49
AGAAAAATCTGATCGGAGCTCTCCTCTTTGATTCAGGGGAGACCGCTGAAGCAACCCGCCTCAAG
CGGACTGCTAGACGGCGGTACACCAGGAGGAAGAACCGGATTTGTTACCTTCAAGAGATATTCTC
CAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCATAGGCTGGAAGAATCATTCCTCGTGGAAG
AGGATAAGAAGCATGAACGGCATCCCATCTTCGGTAATATCGTCGACGAGGTGGCCTATCACGAG
AAATACCCAACCATCTACCATCTTCGCAAAAAGCTGGTGGACTCAACCGACAAGGCAGACCTCCG
GCTTATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCGACC
TCAATCCTGACAATAGCGATGTGGATAAACTGTTCATCCAGCTGGTGCAGACTTACAACCAGCTC
TTTGAAGAGAACCCCATCAATGCAAGCGGAGTCGATGCCAAGGCCATTCTGTCAGCCCGGCTGTC
AAAGAGCCGCAGACTTGAGAATCTTATCGCTCAGCTGCCGGGTGAAAAGAAAAATGGACTGTTC
GGGAACCTGATTGCTCTTTCACTTGGGCTGACTCCCAATTTCAAGTCTAATTTCGACCTGGCAGA
GGATGCCAAGCTGCAACTGTCCAAGGACACCTATGATGACGATCTCGACAACCTCCTGGCCCAGA
TCGGTGACCAATACGCCGACCTTTTCCTTGCTGCTAAGAATCTTTCTGACGCCATCCTGCTGTCT
GACATTCTCCGCGTGAACACTGAAATCACCAAGGCCCCTCTTTCAGCTTCAATGATTAAGCGGTA
TGATGAGCACCACCAGGACCTGACCCTGCTTAAGGCACTCGTCCGGCAGCAGCTTCCGGAGAAGT
ACAAGGAAATCTTCTTTGACCAGTCAAAGAATGGATACGCCGGCTACATCGACGGAGGTGCCTCC
CAAGAGGAATTTTATAAGTTTATCAAACCTATCCTTGAGAAGATGGACGGCACCGAAGAGCTCC
TCGTGAAACTGAATCGGGAGGATCTGCTGCGGAAGCAGCGCACTTTCGACAATGGGAGCATTCCC
CACCAGATCCATCTTGGGGAGCTTCACGCCATCCTTCGGCGCCAAGAGGACTTCTACCCCTTTCTT
AAGGACAACAGGGAGAAGATTGAGAAAATTCTCACTTTCCGCATCCCCTACTACGTGGGACCCCT
CGCCAGAGGAAATAGCCGGTTTGCTTGGATGACCAGAAAGTCAGAAGAAACTATCACTCCCTGG
AACTTCGAAGAGGTGGTGGACAAGGGAGCCAGCGCTCAGTCATTCATCGAACGGATGACTAACT
TCGATAAGAACCTCCCCAATGAGAAGGTCCTGCCGAAACATTCCCTGCTCTACGAGTACTTTACC
GTGTACAACGAGCTGACCAAGGTGAAATATGTCACCGAAGGGATGAGGAAGCCCGCATTCCTGTC
AGGCGAACAAAAGAAGGCAATTGTGGACCTTCTGTTCAAGACCAATAGAAAGGTGACCGTGAAG
CAGCTGAAGGAGGACTATTTCAAGAAAATTGAATGCTTCGACTCTGTGGAGATTAGCGGGGTCG
AAGATCGGTTCAACGCAAGCCTGGGTACCTACCATGATCTGCTTAAGATCATCAAGGACAAGGAT
TTTCTGGACAATGAGGAGAACGAGGACATCCTTGAGGACATTGTCCTGACTCTCACTCTGTTCGA
GGACCGGGAAATGATCGAGGAGAGGCTTAAGACCTACGCCCATCTGTTCGACGATAAAGTGATG
AAGCAACTTAAACGGAGAAGATATACCGGATGGGGACGCCTTAGCCGCAAACTCATCAACGGAA
TCCGGGACAAACAGAGCGGAAAGACCATTCTTGATTTCCTTAAGAGCGACGGATTCGCTAATCGC
AACTTCATGCAACTTATCCATGATGATTCCCTGACCTTTAAGGAGGACATCCAGAAGGCCCAAGT
GTCTGGACAAGGTGACTCACTGCACGAGCATATCGCAAATCTGGCTGGTTCACCCGCTATTAAGA
AGGGTATTCTCCAGACCGTGAAAGTCGTGGACGAGCTGGTCAAGGTGATGGGTCGCCATAAACC
AGAGAACATTGTCATCGAGATGGCCAGGGAAAACCAGACTACCCAGAAGGGACAGAAGAACAGC
AGGGAGCGGATGAAAAGAATTGAGGAAGGGATTAAGGAGCTCGGGTCACAGATCCTTAAAGAGC
ACCCGGTGGAAAACACCCAGCTTCAGAATGAGAAGCTCTATCTGTACTACCTTCAAAATGGACGC
GATATGTATGTGGACCAAGAGCTTGATATCAACAGGCTCTCAGACTACGACGTGGACGCCATCGT
CCCTCAGAGCTTCCTCAAAGACGACTCAATTGACAATAAGGTGCTGACTCGCTCAGACAAGAACC
GGGGAAAGTCAGATAACGTGCCCTCAGAGGAAGTCGTGAAAAAGATGAAGAACTATTGGCGCCA
GCTTCTGAACGCAAAGCTGATCACTCAGCGGAAGTTCGACAATCTCACTAAGGCTGAGAGGGGCG
50
GACTGAGCGAACTGGACAAAGCAGGATTCATTAAACGGCAACTTGTGGAGACTCGGCAGATTAC
TAAACATGTCGCCCAAATCCTTGACTCACGCATGAATACCAAGTACGACGAAAACGACAAACTTA
TCCGCGAGGTGAAGGTGATTACCCTGAAGTCCAAGCTGGTCAGCGATTTCAGAAAGGACTTTCAA
TTCTACAAAGTGCGGGAGATCAATAACTATCATCATGCTCATGACGCATATCTGAATGCCGTGGT
GGGAACCGCCCTGATCAAGAAGTACCCAAAGCTGGAAAGCGAGTTCGTGTACGGAGACTACAAG
GTCTACGACGTGCGCAAGATGATTGCCAAATCTGAGCAGGAGATCGGAAAGGCCACCGCAAAGT
ACTTCTTCTACAGCAACATCATGAATTTCTTCAAGACCGAAATCACCCTTGCAAACGGTGAGATC
CGGAAGAGGCCGCTCATCGAGACTAATGGGGAGACTGGCGAAATCGTGTGGGACAAGGGCAGAG
ATTTCGCTACCGTGCGCAAAGTGCTTTCTATGCCTCAAGTGAACATCGTGAAGAAAACCGAGGTG
CAAACCGGAGGCTTTTCTAAGGAATCAATCCTCCCCAAGCGCAACTCCGACAAGCTCATTGCAAG
GAAGAAGGATTGGGACCCTAAGAAGTACGGCGGATTCGATTCACCAACTGTGGCTTATTCTGTCC
TGGTCGTGGCTAAGGTGGAAAAAGGAAAGTCTAAGAAGCTCAAGAGCGTGAAGGAACTGCTGGG
TATCACCATTATGGAGCGCAGCTCCTTCGAGAAGAACCCAATTGACTTTCTCGAAGCCAAAGGTT
ACAAGGAAGTCAAGAAGGACCTTATCATCAAGCTCCCAAAGTATAGCCTGTTCGAACTGGAGAA
TGGGCGGAAGCGGATGCTCGCCTCCGCTGGCGAACTTCAGAAGGGTAATGAGCTGGCTCTCCCCT
CCAAGTACGTGAATTTCCTCTACCTTGCAAGCCATTACGAGAAGCTGAAGGGGAGCCCCGAGGAC
AACGAGCAAAAGCAACTGTTTGTGGAGCAGCATAAGCATTATCTGGACGAGATCATTGAGCAGA
TTTCCGAGTTTTCTAAACGCGTCATTCTCGCTGATGCCAACCTCGATAAAGTCCTTAGCGCATAC
AATAAGCACAGAGACAAACCAATTCGGGAGCAGGCTGAGAATATCATCCACCTGTTCACCCTCAC
CAATCTTGGTGCCCCTGCCGCATTCAAGTACTTCGACACCACCATCGACCGGAAACGCTATACCT
CCACCAAAGAAGTGCTGGACGCCACCCTCATCCACCAGAGCATCACCGGACTTTACGAAACTCGG
ATTGACCTCTCACAGCTCGGAGGGGATGAGGGAGCTGATGATCCAAAAAAGAAGAGAAAGGTAG
ATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGGGCCcGGTTCCGGTTC
CGGTTCCGGTTCCGGTTCCCAACTCGTGAAGAGTGAACTTGAGGAGAAAAAGTCGGAGCTGCGGC
ACAAATTGAAATACGTACCGCATGAATACATCGAACTTATCGAAATTGCTAGGAACTCGACTCA
AGACAGAATCCTTGAGATGAAGGTAATGGAGTTCTTTATGAAGGTTTATGGATACCGAGGGAAG
CATCTCGGTGGATCACGAAAACCCGACGGAGCAATCTATACGGTGGGGAGCCCGATTGATTACGG
AGTGATCGTCGACACGAAAGCCTACAGCGGTGGGTACAATCTTCCCATCGGGCAGGCAGATGAGA
TGCAACGTTATGTCGAAGAAAATCAGACCAGGAACAAACACATCAATCCAAATGAGTGGTGGAA
AGTGTATCCTTCATCAGTGACCGAGTTTAAGTTTTTGTTTGTCTCTGGGCATTTCAAAGGCAACT
ATAAGGCCCAGCTCACACGGTTGAATCACATTACGAACTGCAATGGTGCGGTTTTGTCCGTAGAG
GAACTGCTCATTGGTGGAGAAATGATCAAAGCGGGAACTCTGACACTGGAAGAAGTCAGACGCA
AGTTTAACAATGGCGAGATCAATTTCCGCTCATAA
4-3-
Amino acid sequence
MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY
PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ
51
LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD
LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDL
LRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW
MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY
VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH
DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG
RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL
AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS
QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLT
RSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV
ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA
YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN
GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI
ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG
YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN
EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG
APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDEGADDPKKKRKVDPKKK
RKVDPKKKRKVGPGSGSGSGSGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMK
VMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEEN
QTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEM
IKAGTLTLEEVRRKFNNGEINFRS*
52
Map of the human plasmid carrying WT.FOK.dCas9
53
Map of the human plasmid carrying Sharky.FOK.dCas9
54
55
pENTRD.Topo.dCas9 Map
56
pDEST26.dCas9 Map
57