Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Design of Guide RNA Sequences
2.2. Vector Construction
2.3. In Vitro Culture of Porcine Fibroblasts and Transfection
2.4. Targeted Deep Sequencing
3. Results and Discussion
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AID | Activation-Induced Cytidine Deaminase |
APOBEC | Apolipoprotein B mRNA Editing, Catalytic Polypeptide-like |
BE | Base Editors |
Cas9 | CRISPR-associated protein 9 |
CHO | Chinese Hamster Ovary |
CRISPR | Clustered Regularly Interspaced Short Palindromic Repeats |
DMEM | Dulbecco’s Modified Eagle Medium |
DNA | Deoxyribonucleic Acid |
DSB | Double Strand Breaks |
PAM | Protospacer Adjacent Motif |
PCR | Polymerase Chain Reaction |
PERV | Porcine Endogenous Retrovirus |
PmCDA1 | Petromyzon marinus Cytidine Deaminase 1 |
RNA | Ribonucleic Acid |
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Target Gene | Direction | Stop Codon | CRISPR Target | Editing Window | Position | PERV-A | PERV-B | PERV-C |
---|---|---|---|---|---|---|---|---|
PERV gag | + | CAA | CCAACGCCTCACGGGGTTGGTGG | CAAC | 705 | o | o | o |
+ | CAG | TTCAGGTTAAGAAGGGACCTTGG | TCAG | 83 | o | o | o | |
+ | GCAGACACTCTTCACAGCCGAGG | CAGA | 780 | x | x | x | ||
+ | CCAGAAAGCCTCAGTGGCCCTGG | CAGA | 1122 | o | o | o | ||
+ | TCAGAGACTGGAAGGGTTACAGG | CAGA | 1185 | o | o | o | ||
+ | CGA | GCGAGAGAGAATTCTGTTAGAGG | CGAG | 804 | o | o | x | |
PERV pol | + | CAA | TCAAGATATACAGTCCTGGTTGG | CAAG | 126 | o | o | x |
+ | CCCAAACCCTAGGACCATGGAGG | CCAA | 1214 | o | o | x | ||
+ | CAG | ACAGTACCCCTTGAGTAGAGAGG | CAGT | 255 | o | o | o | |
+ | GACAGTACACCCTAGAAGACTGG | ACAG | 2105 | o | o | o | ||
+ | CCAGTTCTCTGAGACTCCGGAGG | CAGT | 2148 | o | o | o | ||
+ | CGA | AGCGATGGCTGACGGAGGCACGG | GCGA | 899 | o | o | o | |
+ | TCCGAGATTTGGAATACCTAAGG | CCGA | 2582 | x | o | o | ||
- | CCA | ACCAGTTCCGTTCAGGCGGGAGG | CCAG | 483 | o | o | x | |
- | TCA | CTTCAGTTGAATAACCTGTGGGG | TTCA | 206 | o | o | x | |
- | CTA | TTCTAAGCAGTCCTGTTTGGTGG | TCTA | 761 | o | o | o | |
- | TTCTAGGGTGTACTGTCGTCTGG | TCTA | 2099 | o | o | o | ||
PERV env | + | CAG | AACAGGAAAATATTCAAAAGTGG | ACAG | 581 | x | o | x |
+ | ACCAGGGGTGGTTTGAAGGATGG | CCAG | 1751 | o | o | x | ||
+ | CGA | CCGAGTGTACTACCATCCTGAGG | CGAG | 1308 | x | o | x | |
- | CTA | GTCTATAAGGCGTTTACTACTGG | TCTA | 122 | x | o | x | |
- | CCA | GACCATGACACAGAAATCTTTGG | ACCA | 1274 | x | o | x | |
- | ACCATCCTTCAAACCACCCCTGG | CCAT | 1752 | o | o | x | ||
- | ACCCACTCGTTCTCTAACAAAGG | CCCA | 1883 | x | x | x | ||
- | TCA | CGTCAGAGCAGAAAGCAGGGTGG | GTCA | 1796 | o | o | x | |
- | CTA | TCCTATGCATGTCCCCTTCCCGG | CCTA | 1100 | x | o | x |
Purpose | Strand | Sequence |
---|---|---|
Vector integration confirmation | F | 5′-CCTCGTGCTTTACGGTATCG-3′ |
R | 5′-ATGCTCAAGGGGCTTCATGA-3′ | |
PERV-gag | 1st-F | 5′-CTGGTGGTCTCCTACTGTCG-3′ |
1st-R | 5′-CTCCAAGAGCCAGGATTCGG-3′ | |
2nd-F | 5′-GTCTTGTGCGTCCTTGTCTA-3′ | |
2nd-R | 5′-CGTAAGGATATAGGGCTCCT-3′ | |
PERV-pol | 1st-F | 5′-CCATCACTGTGTTGACCCTC-3′ |
1st-R | 5′-GGTGTAATCTCAGGCAGAAG-3′ | |
2nd-F | 5′-TATACAGTCCTGGTTGGAGC-3′ | |
2nd-R | 5′-ATTGACCTCTCTCAAGTCCT-3′ |
Target Gene | C-to-T Substitution (%) | |
---|---|---|
PERV-gag | Nontreated | 0.22 |
Colony #1 | 63.15 | |
Colony #2 | 1.65 | |
Colony #3 | 52.12 | |
PERV-pol | Nontreated | 0.11 |
Colony #1 | 54.60 | |
Colony #2 | 1.61 | |
Colony #3 | 47.83 |
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Yum, S.-Y.; Jang, G.; Koo, O. Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts. Animals 2021, 11, 3570. https://doi.org/10.3390/ani11123570
Yum S-Y, Jang G, Koo O. Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts. Animals. 2021; 11(12):3570. https://doi.org/10.3390/ani11123570
Chicago/Turabian StyleYum, Soo-Young, Goo Jang, and Okjae Koo. 2021. "Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts" Animals 11, no. 12: 3570. https://doi.org/10.3390/ani11123570
APA StyleYum, S.-Y., Jang, G., & Koo, O. (2021). Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts. Animals, 11(12), 3570. https://doi.org/10.3390/ani11123570