Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System
Abstract
:1. Introduction
2. Results
2.1. Experiment 1: Genome Editing of Two Target Loci through One-Shot Transfection and Subsequent Selection with IB4SAP
2.2. Experiment 2: Genome Editing of Two Target Loci through One-Shot Transfection and Subsequent Selection with IB4SAP
2.3. Experiment 3: Detecting Possible Off-Target Mutations in the Target Sites of Genome-Edited Cells
3. Discussion
4. Materials and Methods
4.1. Cells
4.2. Construction of pCGsap1-Based Vectors for Simultaneous Expression of hCas9 and sgRNA
4.3. Transfection and Selection of Genome-Edited Clones after Targeted Toxin-Based Selection
4.4. SCNT
4.5. Genomic DNA Isolation, PCR, and Sanger Sequencing of Mutated Sites
4.6. WGA Using A Single Blastocyst
4.7. Screening and Detection of Off-Target Sites
4.8. T7E1-Based Assay and Sequencing
4.9. Staining with AF594-IB4 and Detection of Fluorescence
4.10. Immunocytochemistry
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
AF594-IB4 | Alexa Fluor 594-labeled BS-I-B4 isolectin |
α-GalT | α-1,3-galactosyltransferase |
DAPI | 4′,6-Diamidino-2-phenylindole, dihydrochloride |
D-PBS | Dulbecco’s modified phosphate-buffered saline without Ca2+ and Mg2+, pH 7.4 |
EGFP | Enhanced green fluorescent protein |
EndoGalC | Endo-β-galactosidase C |
FACS | Fluorescence-activated cell sorting |
FBS | Fetal bovine serum |
GFP | Green fluorescent protein |
GM | Genetically modified |
h | Humanized |
IB4SAP | Saporin toxin-labeled BS-I-B4 isolectin |
KO | Knockout |
LDLR | Low density lipoprotein receptor |
MPEF | Microminipig embryonic fibroblastic cell |
NGS | Normal goat serum |
NHEJ | Non-homologous end joining |
PEF | Porcine embryonic fibroblastic cell |
PAM | Protospacer adjacent motif |
PCR | Polymerase chain reaction |
PFA | Paraformaldehyde |
PTEN | Deletion of phosphatase and tensin homolog from chromosome 10 |
SCNT | Somatic cell nuclear transfer |
sg | Single guide |
VPA | Valproic acid |
WGA | Whole genome amplification |
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Oligonucleotides (Oligo) Used for Cloning into SapI Site in pCGSap1 | Target Gene | Reference |
---|---|---|
LDLR-oligo: 5′-ACC GGCTG GAAGC ATGAA GTCCA G-3′ 3′-CCGAC CTTCG TACTT CAGGT CAAA-5′ | Porcine LDLR | Sus scrofa breed mixed chromosome 2, Sscrofa10.2 Sequence ID: ref (NC_010444.3) |
p53-oligo: 5′-ACC GGAGG AGTCG CAGTC CGAGC G-3′ 3′-CCTCC TCAGC GTCAG GCTCG CAAA-5′ | Porcine p53 | Sus scrofa breed mixed chromosome 12, Sscrofa10.2 Sequence ID: ref (NC_010454.3) |
PTEN-oligo: 5′-ACC AGATC GTTAG CAGAA ACAAA G-3′ 3′-TCTAG CAATC GTCTT TGTTT CAAA-5′ | Porcine PTEN | Sus scrofa breed mixed chromosome 14, Sscrofa10.2 Sequence ID: ref (NC_010456.4) |
Property | Loci Targeted | LA-1 | LA-2 | LA-3 | Clones LA-4 | LA-5 | LA-6 | LA-7 |
---|---|---|---|---|---|---|---|---|
Direct sequencing of PCR products 1 | GGTA1 LDLR | Normal Normal | Bi-allelic Mono-allelic | Normal Normal | Bi-allelic Bi-allelic | Bi-allelic Mono-allelic | Bi-allelic Bi-allelic | Bi-allelic Mono-allelic |
Expression of α-Gal epitope, as evaluated by cytochemical staining with AF594-IB4 2 | GGTA1 | ++ | − | ++ | − | − | − | − |
Expression of LDLR, as evaluated by immunocytochemical staining using anti-LDLR 2 | LDLR | + | NT | NT | NT | NT | − | NT |
Clones | Locus | Genotyping of Clones by Direct Sequencing of PCR Products and Inserts Sub-Cloned into TA Cloning Vector | Expression of α-Gal Epitope, as Evaluated by Cytochemical Staining with AF594-IB4 | Expression of Protein, as Evaluated by Immunocytochemical Staining Using Antibodies |
---|---|---|---|---|
PPA-1 | GGTA1 | Deletion type (bi-allelic) | − | |
PTEN | Mixture of indel type (bi-allelic) and wild-type | No | Yes | |
p53 | Mixture of deletion (bi-allelic) and replacement (bi-allelic) types | No | ||
PPA-2 | GGTA1 | Deletion type (bi-allelic) | − | |
PTEN | Deletion type (mono-allelic) | No | Yes | |
p53 | Insertion type (bi-allelic) | No |
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Sato, M.; Miyoshi, K.; Nakamura, S.; Ohtsuka, M.; Sakurai, T.; Watanabe, S.; Kawaguchi, H.; Tanimoto, A. Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System. Int. J. Mol. Sci. 2017, 18, 2610. https://doi.org/10.3390/ijms18122610
Sato M, Miyoshi K, Nakamura S, Ohtsuka M, Sakurai T, Watanabe S, Kawaguchi H, Tanimoto A. Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System. International Journal of Molecular Sciences. 2017; 18(12):2610. https://doi.org/10.3390/ijms18122610
Chicago/Turabian StyleSato, Masahiro, Kazuchika Miyoshi, Shingo Nakamura, Masato Ohtsuka, Takayuki Sakurai, Satoshi Watanabe, Hiroaki Kawaguchi, and Akihide Tanimoto. 2017. "Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System" International Journal of Molecular Sciences 18, no. 12: 2610. https://doi.org/10.3390/ijms18122610