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Supplementary information
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Supplementary Table 1.
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Target sequences of EGFP, TK and DP.
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Name of Gene
DNA sequences of crRNA (5’-3’)
1, GACCAGGATGGGCACCACCC
EGFP
2, GGAGCGCACCATCTTCTTCA
TK
GTGCTCTTGCCCGCGAACAT
DP
CCGTGTTCCTCACGTACTCG
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Supplementary Table 2.
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Primers used in this study.
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Name of Gene
Primers (5’-3’)
AGAGGTCTATGCGCGACTAT
ORF81
FAM-AGACACTGAGAGCGTCATCGGTCA-BHQ1
CACATCTTGCCGGTGTACTT
GTTTAGTGAACCGTCAGATCCG
EGFP
GACTTGTACAGCTCGTCCATG
GCTATGCTGGAACTGGTGAT
TK
TGAAGGGCTGCTGCATAAA
GAGTCGATCGTGTCGAAGATG
DP
GTCTTCATGCGCTTCTTGTATTG
TGAAGTAACTGCAGGACGAG
Neo
AATATCACGGGTAGCCCACG
U6 in pX330-puro
GATTCCTTCATATTTGCATATAC
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Supplementary Figure 1.
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CRISPR/Cas9 system used in this study.
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A
Apa LI(9728)
puc ori
U6 promoter
tracRNA
U6 terminator
CBh promoter
Amp r
Apa LI(8482)
Apa LI(7985)
pX330-puro
10046 bp
PURO
Cas9
PGK promoter
poly A
poly A
B
crRNA tracrRNA
H1
bGHpA
CBh
Cas9
Puro
guide RNA
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(A-B). The improved CRISPR system consists of a key protein, Cas9, two designed RNA elements
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forming a duplex (gRNA), and a puromycin resistance gene.
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Supplementary Figure 2.
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Identification of the clonal cells and mutation detection of TK.
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A
B
Puro
Cas9
TK
β-Actin
DP
C
Alignment of DP :
n=30
ATGGACCCCTTCTGCCTCGAGTACGTGAGGAACACGGTCATGCTGGACTGGAAG
ATGGACCCCTTCTGCCTCGAGTACGTGAGGAACACGGTCATGCTGGACTGGAAG
--
control
-2
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(A). After puromycin selection and recovery, representative clones were selected and verified by PCR.
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The sense primer used for detecting TK and DP was U6 primer.
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(B). Representative clones were verified by Western blot with a Cas9 antibody (santa cruze 392740).
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Cells without transfection of any vectors were chosen as control.
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(C). We obtained 30 T-A colonies of targeted fragments amplified from the CyHV-3 infected cells after
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transfection with Cas9-TK. Sequencing data indicated 1 mutation 5 nt from the PAM site (shown in
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green).
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Supplementary Figure 3.
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Standard curve for ORF81 and morphological detection of infected KF-1.
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A
CyHV-3 DNA standard
Cycle Threshold Value
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30
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y = -4.038x + 38.741
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R2 =0.9838
0
1
2
3
Log (Copy Number)
4
5
6
B
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(A). For detecting the viral copy number, the infected cultures were examined by qPCR for the presence
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of CyHV-3 DNA, and a taqman primer set targeting ORF81 was established at 10-fold dilutions. The
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experiments were performed in triplicate.
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(B). KF-1 cells are a useful cell line for productive replication of CyHV-3. The CPE was characterized
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by extensive vacuolation (black arrows).
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