Supplementary Figure 1
NgAgo binds ssDNA guide in a one-guide-faithful manner.
NgAgo-expressing plasmid was transfected to 293T cells and then NgAgo was purified and used in an in vitro plasmid cleavage assay.
The NgAgo derived from the cells co-transfected with target-complementary guide (FW, Lane 4) but not that derived from the cells cotransfected with a random guide (NC, Lane 5) could cause DSBs and linearize the plasmid. The NgAgo derived from the cells without
guide co-delivery could not cleave the target even if the purified NgAgo was later co-incubated with the FW guide (Lane 3). After
NgAgo binds to an ssDNA, it will not swap its bound guide (here is NC) to another free guide (here is FW) at 37℃ (Lane 6).
Representative figure of 3 independent experiments.
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary Figure 2
Guide reloading process at 55℃ impairs endonuclease activity of NgAgo.
NgAgo was purified from E.Coli. and then co-incubated with 5’ phosphorylated ssDNA guide at 37 ℃ or 55℃ for 1 hour or 72 hours.
NgAgo was then subject to a plasmid cleavage assay to test its endonuclease activity. It shows that 55℃ for 1 hour changes the activity
of NgAgo to a nickase and 55℃ for 72 hours completely deprived of its activity. SC, supercoiled; Lin, linearized; OC, open circular.
Representative figure of 3 independent experiments.
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary Figure 3
NgAgo has no activity to cleave linearized target or single strand target.
(a) pACYCDeut-eGFP plasmid was first linearized by BamHI restriction cleavage and then co-incubated with or without the purified
NgAgo preloaded with FW guide in 293T cells. NgAgo was not found to further cleave the target sequence. (b) An 86nt ssDNA coincubated with or without the purified NgAgo preloaded with guide in 293T cells at 37℃ for 8 hours. NgAgo was not found to cleave the
target ssDNA. Representative figures of 3 independent experiments.
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary Figure 4
NgAgo is directed into nucleus by NLS.
The engineered NLS-NgAgo was transfected to Hela cells and it shows that the expressed NLS-NgAgo was compartmented in the
nucleus (DAPI+) and the cells maintained normal morphology.
Scale bar = 100 μm. Representative figure of 20 independent
experiments.
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary Figure 5
T7E1 assay shows the efficiency of NgAgo/gDNA system in cleaving genome.
Forty-seven guides targeting 8 mammalian genome loci were tested for the cleavage efficiency of NgAgo. The percentages of indels
were measured by T7E1 assay. Upper, n = 3; lower, representative figure of 3 independent experiments.
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary Figure 6
T7E1 assay using a 21nt long ssDNA guide derived from G10 to test the effects of nucleotide mismatches on the efficiency of
NgAgo target cleavage.
Mismatches are marked in red. Representative figure of 3 independent experiments.
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary Figure 7
A detailed structure of the donor mRFP-TGA-eGFP DNA fragment.
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary Figure 8
NgAgo has less off-target effect than Cas9 in mammalian cells.
Experimental schematic of investigating off-target genome editing. gDNA and gRNA were designed targeting the same locus of the
genome. A 400bp GFP gene fragment donor (GFP400) without any homologous sequence to the target was co-transfected with either
NgAgo/gDNA or Cas9/gRNA. Thus, the donor could be integrated into any DSBs in the genome. Total genomic DNAs were extracted
from the engineered cells and digested by endonuclease restriction enzymes Bgl II, Sal I, Sac I, Xho I, afl II and Eco47 III. Bgl II
reaction generates a 6.5kb fragment containing the on-target fragment, while other fragments in unknown length are off-targets. (b)
Southern blot analysis detected off-target editing by Cas9 but not by NgAgo. Representative figure of 3 independent experiments.
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary Figure 9
Full-length gel images (Unrelated lanes are marked with cross).
a, for Fig 1a：Nucleic acids associated with NgAgo in E.coli.
b, for Fig 1b: The in vitro plasmid cleavage assay(E.coli.-derived NgAgo).
c, for Fig 1c: The in vitro plasmid cleavage assay(E.coli.-derived NgAgo, guides with or without 5' phosphorylation).
d, for Fig 2a.
e, for Fig 2b.
f, for Fig 2c.
g, for Fig 3a: The in vitro plasmid cleavage assay (293T cell-derived NgAgo).
h, for Fig 3c:western blot（GFP,ACTIN）.
i, for Fig 3d:western blot（GFP,ACTIN）.
j, for Fig 4a: T7E1 (DYRK1A) .
k, for Fig 4b: T7E1 (DYRK1A,EMX1,GRIN2B,GATA4,HBA2).
Nature Biotechnology: doi:10.1038/nbt.3547
l, for Fig 4c: T7E1 (DYRK1A(293T,MCF-7, K562, Hela)).
m, for Fig 4d: mismatches test on 24nt ssDNA guide.
n, for Fig 4e: DYRK1A (NgAgo vs Cas9).
o, for Fig 4f:HBA2,GATA4 (NgAgo vs Cas9).
p, for Supfig 1: The in vitro plasmid cleavage assay (293T cell-derived NgAgo).
q, for Supfig 2: The in vitro plasmid cleavage assay (E. Coli-derived NgAgo).
r, for Supfig 3a: The in vitro linear plasmid cleavage assay.
s, for Supfig 3b: The in vitro ssDNA cleavage assay.
t, for Supfig 5: T7E1 (HBA2,GATA4,GRIN2B,HRES1,APOE).
u, for Supfig 6: mismatches test on 21nt ssDNA guide.
v, for Supplementary fig. 8: Southern blot.
w, for Supplementary fig. 9: A representative experiment for NgAgo/gDNA-mediated genome editing and examination.
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary Figure 10
A representative experiment for NgAgo/gDNA-mediated genome editing and examination (T7E1 assay).
(a) Schematic of experimental design shows the NgAgo/gDNA guides/target and Cas9/gRNA guides/target position, genomic PCR
product and T7EI cleavage positions. Sequences of NgAgo guides: G5：5'P-CCTACCAGAATCGCCCAGTGGCTG-3'
G10：5'P-CCAAAGTCCAAGGTATTAGCAGCC-3'
Sequence of Cas9 guide: sg-DYRK1A: 5'-TAGCAGCCACTGGGCGATTC-3'
Genomic PCR primers: DY 001 F：5’-GAAGCTCCTACACAGGTCACTG-3’
DY 705 R ：5’-TTGCCCTCTTGTAGCGGTT-3’
(b) T7EI results for NgAgo/gDNA(G5 and G10) and Cas9/gRNA(sg-DYRK1A).
Nature Biotechnology: doi:10.1038/nbt.3547
Index of combined PDF Supplementary Data:
supplementary Note 1: A general protocol of NgAgo/gDNA-mediated genome
editing and examination (T7E1 assay).
Supplementary tables.
A general protocol of NgAgo/gDNA-mediated genome editing and examination (T7E1
assay)
1. Cell culture
293T cells are maintained in high-glucose DMEM (HyClone) supplemented with 10% FBS
(HyClone) and penicillin/streptomycin at 37°C with 5% CO2 incubation. Cells are seeded to
24-well plate (Costar) with 60% confluence 8 hours before transfection.
Thirty minutes before
transfection, cells are washed twice with PBS and medium is changed to high-glucose DMEM
medium containing 2% FBS.
2. Transfection
2-1 NLS-NgAgo expressing plasmid is extracted with Wizard® Plus SV Minipreps DNA
Purification System (Promega), and is adjusted to 100 ng/μl in 0.5x TE buffer (5 mM
Tris-HCl, 0.5 mM EDTA，pH 8.0).
2-2 5’-phosphorylated ssDNA guides are dissolved to 100 ng/μl in 0.5x TE buffer (PH 8.0).
For each well of a 24-well plate, 200-250 ng NLS-NgAgo plasmid and 100-300 ng guides
are diluted in 50 μl Opti-MEM (Gibco); 1.25 μl lipofectamine 2000 is diluted in 50 μl
Opti-MEM.
Incubate the DNA mix and lipofectamine mix for 5 min.
2-3 Combine the DNA mix and lipofectamine mix with gentle pipetting and incubate for 20 min.
The DNA/lipo mixture is then added into each well of cells.
*Since NgAgo follows “one-guide faithful” rule, i.e. guide can only be loaded when NgAgo
protein is in the process of expression, to improve the efficiency of gDNA loading to
NgAgo, multiple transfection of gDNA can be conducted (e.g., 24 hours after the primary
transfection)
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Nature Biotechnology: doi:10.1038/nbt.3547
*As stated below, cells will be harvested 48-60 hours after transfection. 90% confluence of the
cells on harvesting is ideal.
Cell overplating significantly weakens the efficacy of genome
editing. Taking HDR as an example, it occurs only during S and G2 phases.
3. Genomic DNA extraction
3-1 48-60 hours after transfection, cells are harvested by trypsin digestion.
Four wells of cells
are combined into a 1.5 ml EP tube.
3-2 For genomic DNA extraction, 500 μl of cell lysate buffer (50 mM Tris，100 mM EDTA，0.5%
SDS，pH 8) and 10 μl proteinase K (10 mg/ml）are added into each tube and mixed gently
and sufficiently.
Bathe the tubes at 55℃ for 2 hours.
3-3 200 μl Tris-Phenol and 200 ul trichloromethane are added into each tube and mixed gently
and sufficiently. After incubation for 5 min, samples are spun at 12,000 rpm for 15 min to
separate aqueous phase from Phenol phase.
3-4 Carefully collect the aqueous phase into a clean EP tube.
3-5 Repeat the Steps 3-3 and 3-4 once and pool the aqueous phase.
3-6 Add 500 μl trichloromethane into the collected aqueous phase, mix gently and sufficiently,
stay for 5 min, and then spin the sample at 12,000 rpm for 15 min to separate aqueous phase
from Phenol phase. Carefully remove the aqueous phase into a clean EP tube.
3-7 Repeat the Steps 3-6 once.
3-8 Add 900 μl EtOH to the collected aqueous phase and incubate at -20°C for 30 min.
3-9 Centrifuge the sample at 12,000 rpm for 10 min, and then the DNA sediment is washed with
500 μl 75% EtOH thrice.
3-10 Air-dry the DNA sediment, add 50 μl 0.5 x TE and then adjust the genome DNA to 100
ng/μl for later use.
4. PCR Amplification (a brief example)
4-1 For 20 ul reaction system：
Genomic DNA
1 μl
Primer 1: (10 umol/ μl）
0.5 μl
Primer 2: (10 umol/ μl）
0.5 μl
2
Nature Biotechnology: doi:10.1038/nbt.3547
2 x Taq PCR starmix with loading dye（GenStar）
10 μl
H2O
8 μl
4-2 PCR program：
96℃ 3min
94℃ 30s
57℃ 30s
72℃ 20s
30
cycles
72℃ 5min
5. T7EI reaction
5-1 PCR products are purified with a PCR purification kit.
5-2 Annealing: PCR products 150 ng, 10 x NEBuffer2 1 μl, with ddH2O to a final volume of 9.6
μl.
95℃
5 min
95~85℃
-2°C/second
85~25℃
-0.1°C/second
4℃
1 hour
5-3 Mix the annealing product 9.6 μl with T7EI 0.4 μl and incubated the mixture at 37℃ for 30
min.
6. PAGE analysis
6-1 Equipment: Bio-Rad Mini-PROTEAN, 1 mm plate and comb.
6-2 PAGE gel preparation:
30% Arc-Bis(19:1) solution with 4M Urea
1 ml
5 x TBE
1.5 ml
ddH2O
4 ml
10% APS
85 μl
TEMED
3.8 μl
6-3 Run pre-electrophoresis at 140v for 30 min.
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Nature Biotechnology: doi:10.1038/nbt.3547
6-4 Mix the 10 μl T7EI reaction product with 2 μl 6x loding buffer (Biolab) and ran
electrophoresis at 140v for 30min.
7. Silver staining
7-1 Peel the Gel off from the plate and directly put it into Fix solution. Shake gently for 30
min.
7-2 Wash with Ultra-pure MiliQ water for 4 min each time thrice in dish.
7-3 Add ice-chilled staining solution and shake slowly for 25 min.
7-4 Wash with MiliQ water thrice (10 seconds each time).
7-5 Add ice-chilled developing solution and shake slowly till bands appear clearly.
7-6 Decant the developing solution and then add Fix solution to terminate reaction.
*Fix solution：10% acetic acid in MiliQ water
Staining solution：0.1 g Silver nitrate and 150 μl formaldehyde into 100 ml MiliQ water.
Developing solution：6 g sodium carbonate, 300 μl formaldehyde and 13 μl 30% sodium
thiosulfate into 200 ml MiliQ water.
8. A representative experiment (Supplementary fig. 9.)
4
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary tables
Supplementary Table 1, Guides used in this study
Guide
Sequence
Gene
FWG
5'P-TGCTTCAGCCGCTACCCCGACCAC-3'
GFP
RVG
5'P-GTGGTCGGGGTAGCGGCTGAAGCA-3'
GFP
NCG
5'P-CCGCCCCGAGTTCAAGGTGGAGCG-3'
random
G1
5'P-CGGTAAACTGCCCACTTGGCAGTA-3'
CMV
G2
5'P-CCAAGTAGGAAAGTCCCATAAGGT-3'
CMV
G3
5'P-AAGGGCGAGGAGCTGTTCACCGGG-3'
GFP
G4
5'P-GTGGTCGGGGTAGCGGCTGAAGCA-3'
GFP
G5
5'P-CCTACCAGAATCGCCCAGTGGCTG-3'
DYRK1A
G6
5'P-CAGCCACTGGGCGATTCTGGTAGG-3'
DYRK1A
G7
5'P-ATCGCCCAGTGGCTGCTAATACCT-3'
DYRK1A
G8
5'P-AGGTATTAGCAGCCACTGGGCGAT-3'
DYRK1A
G9
5'P-GGCTGCTAATACCTTGGACTTTGG-3'
DYRK1A
G10
5'P-CCAAAGTCCAAGGTATTAGCAGCC-3'
DYRK1A
G11
5'P-ATACCTTGGACTTTGGACAGAATG-3'
DYRK1A
G12
5'P-CATTCTGTCCAAAGTCCAAGGTAT-3'
DYRK1A
G13
5'P-GCTCCATTCTGTCCAAAGTCCAAG-3'
DYRK1A
G14
5'P-AGACGGTCAAATTAACGTCCATAG-3'
DYRK1A
G15
5'P-CTGCTCCTCTTGGTTGGTCAGGCA-3'
DYRK1A
G16
5'P-TGCCTGACCAACCAAGAGGAGCAG-3'
DYRK1A
G17
5'P-CTCCTCTTGGTTGGTCAGGCACTG-3'
DYRK1A
G18
5'P-CAGTGCCTGACCAACCAAGAGGAG-3'
DYRK1A
G19
5'P-CGCTGTCCACCTTCCAGCAGATGT-3'
ACTIN
G20
5'P-ACATCTGCTGGAAGGTGGACAGCG-3'
ACTIN
G21
5'P-CAGCAAGCAGGAGTATGACGAGTC-3'
ACTIN
G22
5'P-GACTCGTCATACTCCTGCTTGCTG-3'
ACTIN
G23
5'P-GTCCGGCCCCTCCATCGTCCACCG-3'
ACTIN
G24
5'P-CGGTGGACGATGGAGGGGCCGGAC-3'
ACTIN
5
Nature Biotechnology: doi:10.1038/nbt.3547
G25
5'P-CCCTCCATCGTCCACCGCAAATGC-3'
ACTIN
G26
5'P-AGCATTTGCGGTGGACGATGGAGG-3'
ACTIN
G27
5'P-CCCACGAGGGCAGAGTGCTGCTTG-3'
EMX1
G28
5'P-CAAGCAGCACTCTGCCCTCGTGGG-3'
EMX1
G29
5'P-GCCAATGGGGAGGACATCGATGTC-3'
EMX1
G30
5'P-GACATCGATGTCCTCCCCATTGGC-3'
EMX1
G31
5'P-TGTCACCTCCAATGACTAGGGTGG-3'
EMX1
G32
5'P-CCACCCTAGTCATTGGAGGTGACA-3'
EMX1
G33
5'P-GCAACCACAAACCCACGAGGGCAG-3'
EMX1
G34
5'P-CTGCCCTCGTGGGTTTGTGGTTGC-3'
EMX1
G35
5'P-TGCTGGCCAGGCCCCTGCGTGGGC-3'
EMX1
G36
5'P-GCCCACGCAGGGGCCTGGCCAGCA-3'
EMX1
G37
5'P-GAGATGGCGCCTTCCTCTCAGGGC-3'
HBA2
G38
5'P-GCGCCTTCCTCTCAGGGCAGAGGA-3'
HBA2
G39
5'P-CTCTTCTCTGCACAGCTCCTAAGC-3'
HBA2
G40
5'P-GGCGCCCGCGCCGTGCATGAAGGC-3'
GATA4
G41
5'P-AGCTCCGGTGGGGCCGCGTCTGGT-3'
GATA4
G42
5'P-GGTCCCTGGCGGCCGCCGCCGCCG-3'
GATA4
G43
5'P-GATAAGGTCCTTGAATTGCAGTAT-3'
GRIN2B
G44
5'P-TTGCAGGGAGTCGACGAGTTGAAG-3'
GRIN2B
G45
5'P-ATGAATGAGACCGACCCAAAGAGC-3'
GRIN2B
G46
5'P-GCGGGGCCGGGCCTGGGCTGCGGG-3'
HRES1
G47
5'P-ACCGTAGGTTTCGGACATGGCCGT-3'
HRES1
G48
5'P-CTCCACCCTCCGTCCGGCCGCGAC-3'
HRES1
G49
5'P-CGCGTGCGGGCCGCCACTGTGGGC-3'
APOE
G50
5'P-CATGGCCTGCACCTCGCCGCGGTA-3'
APOE
G51
5'P-GCCTCAAGAGCTGGTTCGAGCCCC-3'
APOE
G52
5'P-AGAAGGTATACACGTCGGAAGAAT-3'
target
6
Nature Biotechnology: doi:10.1038/nbt.3547
Supplementary table 2, Primers used in the T7E1 assay
Primer
Sequence
DYRK1A test F
5’-GTTCTTTCAGGTGCGTCA-3'
DYRK1A test R
5’-GGGACTCTTCTCTATCAGCC-3'
HBA2 test F
5’-ACGGCTCTGCCCAGGTTA-3'
HBA2 test R
5’-CATTGTTGGCACATTCCG-3'
GATA4 test F
5’-CCCCTTTGATTTTTGATCTTCG-3'
GATA4 test R
5’-TGTGCAGGACCGGGCTGT-3'
GRIN2B test F
5’-CAGGAGGGCCAGGAGATTTG-3'
GRIN2B test R
5’-TGAAATCGAGGATCTGGGCG-3'
EMX1 test F
5’-CCATCCCCTTCTGTGAATGT-3'
EMX1 test R
5’-GGAGATTGGAGACACGGAGA-3'
APOE test F
5’-GGAACTGGAGGAACAACTGAC-3'
APOE test R
5’-TCGGCGTTCAGTGATTGT-3'
HRES1 test F
5’-ATGCGCTGTGCACAGCGC-3'
HRES1 test R
5’-TCAGGGAAATCGGGACTCAGC-3'
β-ACTIN test F
5’-CACGAAACTACCTTCAACTCC-3'
β-ACTIN test R
5’-GACTTCCTGTAACAACGCATC-3'
7
Nature Biotechnology: doi:10.1038/nbt.3547
Length
584BP
577BP
705BP
696BP
639BP
680BP
676BP
700BP
Supplementary table 3, Oligos, plasmids and constructs used in this study
plasmid
oligos
NgAgo-6
P-1 F
NgAgo-6P-1
NgAgo-6
P-1 R
FLAG Hid
Ⅲ F
FLAG-NgAgo-HA
-pcDNA3.1
HA Bamh
ⅠR
NLS-NgA
go F1
NgAgo-N
LS-Hind
Ⅲ F2
NLS-NgAgo-red
NgAgo-B
am R
NLS-NgAgo-pcD
NA3.1
NgAgo-N
LS-Hind
Ⅲ F2
NgAgo-B
am-TGAR
SpCas9Hind - F
SpCas9-pcDNA3
.1
SpCas9Bam-R
pACY600
-Hind F
pACY268
3-Bam R
GFP-Hin
d R
pACYCDuet-GFP
CMV-Bam
F
sequence
5’-GAAGATCTACA
GTGATTGACCTCGA
TTCG-3’
5’-CCGCTCGAGCT
AGAGGAATCCGACA
TTAGACTCG-3’
5’-CCCAAGCTTGC
CACCATGGATTACA
AGGATGACGACGAT
AAGACAGTGATTGA
CCTCGATTCG-3’
5’-CGGGATCCTTA
AGCGTAATCTGGAA
CATCGTATGGGTAG
AGGAATCCGACATT
AGACTCG-3’
5’-CCAAAAAAGAA
GAGAAAGGTAGCCA
CAGTGATTGACCTC
GATTCG-3’
5’-CCCAAGCTTGC
CACCATGGTGCCAA
AAAAGAAGAGAAAG
GTAGCC-3’
5’-CGGGATCCCGG
AGGAATCCGACATT
AGACTCG-3’
5’-CCCAAGCTTGC
CACCATGGTGCCAA
AAAAGAAGAGAAAG
GTAGCC-3’
5’-CGGGATCCtca
GAGGAATCCGACAT
TAGACTCG-3’
5’-CCCAAGCTTGC
CACCATGGACTATA
AGGACCACGACG-3
’
5’-CGGGATCCTCA
CTTTTTCTTTTTTG
CCTGGC-3’
5’-CCCAAGCTTAA
CGACCCTGCCCTGA
AC-3’
5’-CGCGGATCCGG
GCATGACTAACATG
AGAATTAC-3’
5’-CCCAAGCTTGG
GGGACTTGTACAGC
TCGTCC-3’
5’-CGCGGATCCTA
GTTATTAATAGTAA
TCAATTACG-3’
8
Nature Biotechnology: doi:10.1038/nbt.3547
template
backbone
restrictio
n sites
genomic DNA
of
Natronobact
eriumgregor
yi SP2
pGEX6P-1
BamhI,Bgl
Ⅱand XhoI
genomic DNA
of
Natronobact
eriumgregor
yi SP2
pcDNA3.1
/Hygro(+
)
HindⅢ and
BamhI
genomic DNA
of
Natronobact
eriumgregor
yi SP2
pDsRed
MonomerN1
HindⅢ and
BamhI
NLS-NgAgo-r
ed
pcDNA3.1
/Hygro(+
)
HindⅢ and
BamhI
pX330-U6-Ch
imeric_BB-C
Bh-hSpCas9
pcDNA3.1
/Hygro(+
)
HindⅢ and
BamhI
pACYCDuet-1
EGFP-N1
without MCS
BamhI and
HindⅢ
sgRNA F
sgRNA(DYRK1A)
sgRNA R
sgRNA1 F
sgRNA(CMV)
sgRNA1 R
sgRNA2 F
sgRNA(GFP)
sgRNA2 R
sgRNA3 F
sgRNA(HBA2)
sgRNA3 R
sgRNA4 F
sgRNA(GATA4)
sgRNA4 R
5’-CACCGGCCCAG
TGGCTGCTAATACC
T-3’
5’-AAACAGGTATT
AGCAGCCACTGGGC
C-3’
5’-CACCGGCTGGG
CATAATGCCAGGCG
G-3’
5’-AAACCCGCCTG
GCATTATGCCCAGC
C-3’
5’-CACCGGCTCGT
GACCACCCTGACCT
A-3’
5’-AAACTAGGTCA
GGGTGGTCACGAGC
C-3’
5’-CACCGGGAGAT
GGCGCCTTCCTCTC
A-3’
5’-AAACTGAGAGG
AAGGCGCCATCTCC
C-3’
5’-CACCGGGGCGC
CCGCGCCGTGCATG
A-3’
5’-AAACTCATGCA
CGGCGCGGGCGCCC
C-3’
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Nature Biotechnology: doi:10.1038/nbt.3547
phU6-gRN
A
BbvII
phU6-gRN
A
BbvII
phU6-gRN
A
BbvII
phU6-gRN
A
BbvII
phU6-gRN
A
BbvII