Generation of A Triple Insert Live Avian Herpesvirus Vectored Vaccine Using CRISPR/Cas9-Based Gene Editing
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viruses and Cell Culture
2.2. Construction of sgRNAs and Donor Plasmids
2.3. Generation of Recombinant HVT-VP2-gDgI-HA
2.4. Western Blot Analysis
2.5. Indirect Immunofluorescence Analysis (IFA)
2.6. Stability of the Inserted Genes in the Recombinant Viruses
2.7. In Vitro Growth Kinetics
3. Results
3.1. Generation of Triple Insert HVT Recombinant HVT-VP2-gDgI-HA
3.2. Characterization of Recombinant HVT-VP2-gDgI and HVT-VP2-gDgI-HA
3.3. Stability of Recombinant HVT-VP2-gDgI-HA
4. Discussion
Author Contributions
Funding
Conflicts of Interest
References
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sgRNA | Target Sequences | PAM | Gene Locus |
---|---|---|---|
UL45/46 | GAGATCGAGTGCCGCATCAC | CGG | Between UL45 & UL46 |
HVT65/66 | GGGAAACTAAATGTTCATAG | AGG | Between HVT65 and HVT66 |
US2 | ACACAAATTGCGTTTAGGTG | GGG | US2 |
sg-A | GAGATCGAGTGCCGCATCAC | CGG | sgA |
sg-B | GAGATCGAGTTCGGCTAGAC | CGG | sgB |
sg-C | GAGAGAGTGTGGCGACTCTG | CGG | sgC |
Primer | Sequences |
---|---|
SfiIx2-F | CTAGCAAGGCCGCCTAGGCCGGCGCGCCGTTAAACGGCCATTATGGCCGTTT |
SfiIx2-R | AAACGGCCATAATGGCCGTTTAACGGCGCGCCGGCCTAGGCGGCCTTG |
sgB-LoxN-F | CATGGGAAGTCGAGTTCGGCTAGACCGGATAACTTCGTATAAGGTATACTATACGAAGTTATTTAATTAAATAACTTCGTATAAGGTATACTATACGAAGTTATGGCCGCCTAGGCCGGCGCGCCGTTTAAACGGCCATTATGGCCGAAGTCGAGTTCGGCTAGACCGGCA |
sgB-LoxN-R | TATGCCGGTCTAGCCGAACTCGACTTCGGCCATAATGGCCGTTTAAACGGCGCGCCGGCCTAGGCGGCCATAACTTCGTATAGTATACCTTATACGAAGTTATTTAATTAAATAACTTCGTATAGTATACCTTATACGAAGTTATCCGGTCTAGCCGAACTCGACTTCC |
sgC-Lox2272-F | CATGGGAGAGAGTGTGGCGACTCTGCGGATAACTTCGTATAAAGTATCCTATACGAAGTTATTTAATTAAATAACTTCGTATAAAGTATCCTATACGAAGTTATGGCCGCCTAGGCCGGCGCGCCGTTTAAACGGCCATTATGGCCGAGAGAGTGTGGCGACTCTGCGGCA |
sgC-Lox2272-R | TATGCCGCAGAGTCGCCACACTCTCTCGGCCATAATGGCCGTTTAAACGGCGCGCCGGCCTAGGCGGCCATAACTTCGTATAGGATACTTTATACGAAGTTATTTAATTAAATAACTTCGTATAGGATACTTTATACGAAGTTATCCGCAGAGTCGCCACACTCTCTCC |
HA-F * | CGAGCGGCCGCATGGAAGCACTATCACTGATAACTATAC |
HA-R | GCGGCGGCCGCTTATATACAAATGTTGCATCTGCAAGA |
Primer | Sequence |
---|---|
UL45F | TACCGTTATATGTCAGCGACCCA |
UL46R | CTCCGACAACCAAATACTTTCATGA |
HVT65 F | TCGCTATGCAAAGAGATGCGTG |
HVT66 R | CGTCTGCGATAACTACGCCT |
US2F | CTGTGATACACTTGGGAGCC |
US2R | GACGTTTCCGATCTTCCACA |
VP2 5R | GTGCATGACCGTGCTGATTC |
VP2 3F | CGTCTTGGCATCAAGACCGT |
gDI 5R | ACGACAGGCACATTAGCTGGACC |
gDI 3F | TAGTTACTGTGCCTTCTAGTTGCCAG |
HA 5R | GCTCTGTGTGGAGCAGTTCT |
HA 3F | TGAAGAGAGCGTTGGGTTCC |
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Tang, N.; Zhang, Y.; Sadigh, Y.; Moffat, K.; Shen, Z.; Nair, V.; Yao, Y. Generation of A Triple Insert Live Avian Herpesvirus Vectored Vaccine Using CRISPR/Cas9-Based Gene Editing. Vaccines 2020, 8, 97. https://doi.org/10.3390/vaccines8010097
Tang N, Zhang Y, Sadigh Y, Moffat K, Shen Z, Nair V, Yao Y. Generation of A Triple Insert Live Avian Herpesvirus Vectored Vaccine Using CRISPR/Cas9-Based Gene Editing. Vaccines. 2020; 8(1):97. https://doi.org/10.3390/vaccines8010097
Chicago/Turabian StyleTang, Na, Yaoyao Zhang, Yashar Sadigh, Katy Moffat, Zhiqiang Shen, Venugopal Nair, and Yongxiu Yao. 2020. "Generation of A Triple Insert Live Avian Herpesvirus Vectored Vaccine Using CRISPR/Cas9-Based Gene Editing" Vaccines 8, no. 1: 97. https://doi.org/10.3390/vaccines8010097
APA StyleTang, N., Zhang, Y., Sadigh, Y., Moffat, K., Shen, Z., Nair, V., & Yao, Y. (2020). Generation of A Triple Insert Live Avian Herpesvirus Vectored Vaccine Using CRISPR/Cas9-Based Gene Editing. Vaccines, 8(1), 97. https://doi.org/10.3390/vaccines8010097