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Supplementary Data
CRISPR/Cas9 mediated somatic and germline gene correction to restore
hemostasis of hemophilia B mouse
Cong Huai1*, Ph.D.; Chenqiang Jia1*, M.S.; Ruilin Sun2, Ph.D.; Peipei Xu1, B.S.; Taishan
Min1, B.S.; Qihan Wang1, M.S.; Chengde Zheng1, M.S.; Hongyan Chen1†, Ph.D. & Daru
Lu1†, Ph.D.
1 Key Laboratory of Genetic Engineering, MOE Key Laboratory of Contemporary
Anthropology, Fudan University School of Life Sciences, Shanghai 200438, China
2 Shanghai Research Center for Model Organisms, Shanghai 201210, China
* These authors contribute equally to this work
Correspondence and requests for materials should be addressed to Prof. Daru Lu.
Address: Room C613, Building for School of Life Sciences, Fudan University, No. 2005
Songhu Rd, Shanghai, China
Telephone/Fax: 86-21-51630619
Email: [email protected]
Supplementary Fig. 1 All three sgRNAs used in HTV treatment are effective in
DNA binding and cleavage
a Schematic diagram shows the relative location of the absent DNA (red line) and
sgRNA binding sites. Yellow arrows represent the binding direction of each sgRNA,
pink boxes represent the PAM region. b In vitro DNA cleavage activity of each
sgRNA by RGEN assay. M, DNA ladder. c T7E1 assay performed on genome DNA
extracted from Cas9-sgRNA transfected 293T cells. The frequency of Cas9-induced
indels is indicated as a “%” below each lane.
Supplementary Fig. 2 In vivo gene correction of adult HB mice through
hydrodynamics-based DNA transfection of Cas9 components
a Detection resolution of unlabeled probe by high-resolution melting analysis. b-c
Normalized melting peak of unlabeled probe hybridization performed on hepatic
cDNA obtained from HR1 or HR2 sgRNA-treated mice. d Amplified fragment length
polymorphism assay performed on hepatic cDNA obtained from mice treated as
indicated.
Supplementary Fig. 3 Detection of off-target gene-editing
a Sequence and location of the top 5 potential off-target binding sites of HR3 sgRNA.
The difference between the HR3 binding sequence and the off-target sequence is
highlighted by bold and boxed font. b HRMA of the five potential off-target sites
performed on the microinjected embryos. c Concentration and activity in plasma of
mice treated as indicated.
Supplementary Table 1 Primers used for F9 amplification, real-time PCR and off-
target detection
Primer name Sequence (5’ – 3’)
F9LF CAGTGAAGCCAACCAGACTG
F9LR CAGTTGACGTACCGGGAAAC
F9SF GGAGACAGGCTTCCATTCTTC
F9SR TTCACCCCAGCTAATAATGC
F9 qPCR F TTGAGGCATTCTGTGGAGGT
F9 qPCR R CCAGCAAGGCAATGTCATGA
mActb qPCR F AGTGTGACGTTGACATCCGT
mActb qPCR R GCAGCTCAGTAACAGTCCGC
Unlabeled probe GATCCTTCACACGAATCTTTGCCTCCGG
Hybri-probe DIG-CCACTATCTCCTTCACACGAAT
OFT1-F TCGGTCAACTGTTTACCACTG
OFT1-R GTGGGGCAGGTAGGAGAAAT
OFT2-F CAAACGTAGGCTGAGCACTG
OFT2-R CTTGACCGAGCCTTCCAAAG
OFT3-F GCATGGGTTCAGAGGGCATA
OFT3-R CTGTCCCAGCTCTCTAACCC
OFT4-F AGACTAAACTACAGACACCACGA
OFT4-R GTTCCCTTTTGCCTTGGTGA
OFT5-F CTCCTGACTGAGTTGGGAAC
OFT5-R AGACCCTTGCTTCCTCGTAG
Supplementary Table 2 Treatment effect of Cas9 mediate germline gene therapy
(Based on tail DNA form born mice)
Bornrate
DNA correctionEfficiency
Effective Treatment
Gene Rpaired Sampleshomozygot
e/hemizygote
hetero-zygote
mosaic
mRNA11.7%
(6)36.1%
66.7%(4)
0 0 100%
Cas9-wt41.6%(17)
59.2%82.4%(14)
42.8%(6)
21.4%(3)
35.7%(5)
Cas9-wt, wild type SpCas9 protein. Cas9-HF, high-fidelity SpCas9 protein. All
embryos that can detected to carry corrected gene by Sanger sequencing is signed as
“Gene Repaired Samples”. The treatment effective rate is determined by hemostasis
of born mice. The amount of each type of mice is label in brackets.