Concurrent Gene Insertion, Deletion, and Inversion during the Construction of a Novel Attenuated BoHV-1 Using CRISPR/Cas9 Genome Editing
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viruses and Cell Lines
2.2. Construction of sgRNA and Donor Plasmids
2.3. Screening of Cell Lines for Gene Editing
2.4. The Verification of Sgrna Editing Efficiency on Bohv-1
2.5. Knock Out BoHV-1 gE Gene by CRISPR/Cas9
2.6. Identification of Recombinant Viruses
2.7. Determination of Recombinant Virus Replication Kinetics
2.8. Off-Target Detection and Morphological Verification of Virus Particles
2.9. Mouse Immunization and Determination of Virus Neutralizing Antibodies
2.10. Statistical Analysis
3. Results
3.1. The Construction of Donor Plasmids
3.2. Screening of Cell Lines for Bohv-1 Gene Editing
3.3. sgRNA Editing Efficiency Screening and Identification of Bohv-1 gE/EGFP+ Recombinant Virus
3.4. EGFP Was Further Removed from Recombinant BoHV-1 gE/EGFP+ by sgRNA of EGFP
3.5. Double sgRNAs Editing Increases HR Efficiency
3.6. Identification of Mutant Viruses In Vitro and Serological Analysis
3.7. Stability of Mutant Viruses
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primers | Nucleotide Sequences (5′-3′) | Genome Position | Restriction Sites |
---|---|---|---|
LgE-F | aagcttTGCTCTTCTCCATCGCCCATC | 120663–120683 | HindIII |
LgE-R | ggtaccCATTGCCAAATGCCCTTTTCGA | 121695–121716 | KpnI |
EGFP-F | ggtaccATGGTGAGCAAGGGCGA | 1–17 | KpnI |
EGFP-R | ggatccCTTGTACAGCTCGTCC | 702–717 | BamHI |
R1gE-F | ggatccAGTCGTTACTTCGGACCGTTTGGTGC | 122847–122872 | BamHI |
R1gE-R | gaattcTCAGCGCCTCGATAGTTTTCGTTGAC | 123569–123594 | EcoRI |
R2gE-F | ggatccTCACCATCGAGGACGCGCCGGCCAGCGCAGA | 123663–123693 | BamHI |
R2gE-R | gaattcCGAATCCTCGGCCGGCCCGAATCCCCTCCTT | 124332–124362 | EcoRI |
R3gE-F | ggatccCTCAAGTCCATCCTCCGCTAG | 123421–123441 | BamHI |
R3gE-R | gaattcGCCCTTGTCATATTTTTTTAA | 124486–124506 | EcoRI |
BoHV-gE F | CGCCGGGTTGTTAAATGGGTCTCG | 121573–121596 | |
BoHV-gE R | CGGGCGCGTCCTCGATGGTG | 123664–123683 | |
px459-EGFP-sgRNA1 F | caccgGTCGCCCTCGAACTTCACCT | 335–354 | BbsI |
px459-EGFP-sgRNA1 R | aaacAGGTGAAGTTCGAGGGCGACc | 335–354 | BbsI |
px459-EGFP-sgRNA2 F | caccgGTGGTTGTCGGGCAGCAGCA | 581–600 | BbsI |
px459-EGFP-sgRNA2 R | aaacTGCTGCTGCCCGACAACCACCc | 581–600 | BbsI |
px459-EGFP-sgRNA3 F | caccgGTTGGGGTCTTTGCTCAGGG | 620–639 | BbsI |
px459-EGFP-sgRNA3 R | aaacCCCTGAGCAAAGACCCCAACc | 620–639 | BbsI |
px459-gE-sgRNA1 F | caccgCGGCGACGAGGAGACGCAGTTGG | 122217–122239 | BbsI |
px459-gE-sgRNA1 R | aaacCCAACTGCGTCTCCTCGTCGCCGc | 122217–122239 | BbsI |
px459-gE-sgRNA2 F | caccgCGCCGATGAGCCGGTCGTACAGG | 122190–122212 | BbsI |
px459-gE-sgRNA2 R | aaacCCTGTACGACCGGCTCATCGGCGc | 122190–122212 | BbsI |
px459-gE-sgRNA3 F | caccgCGAGCCCGGGGTTTCGGTCGCGG | 121802–121824 | BbsI |
px459-gE-sgRNA3 R | aaacCCGCGACCGAAACCCCGGGCTCGc | 121802–121824 | BbsI |
px459-gE-sgRNA4 F | caccgCCACGTCGGTGAAGCACTCGCGG | 121977–121999 | BbsI |
px459-gE-sgRNA4 R | aaacCCGCGAGTGCTTCACCGACGTGGc | 121977–121999 | BbsI |
UL53 F | CACTGAGACCGGCATTTTA | 2971–2989 | |
UL53 R | CGAAGAGTTTATTGCTGAC | 4376–4394 | |
UL47 F | ACTTGGGTCTACACGGGATTTA | 11902–11923 | |
UL47 R | TTGTCCTGCTTGTGCTTGAACG | 14783–14804 | |
UL44 F | GACGACTACGAAAACTAC | 16459–16476 | |
UL44 R | GACCACGAAAGCACAAAA | 18318–18335 | |
UL27 F | CAGTTTTTTTGCTTCGCATCCG | 55179–55200 | |
UL27 R | TTTTGCATTACTTTTGGGGTCA | 58536–58557 | |
UL23 F | AAAAACGGCACGTCTTCAGCTC | 62955–62976 | |
UL23 R | ACCACCATTTCCCACTCTTCGA | 64582–64603 | |
UL22 F | GACCCCAGTTGTGATGAATGCA | 63995–64016 | |
UL22 R | GCCGTCGGACAGTGAGTATGAG | 67196–67217 | |
US4 F | CAGATGCTGACCTTTGACTTTC | 117119–117140 | |
US4 R | GTTTAACTCGCAATAGACACGC | 118755–118776 | |
US6 F | GACGCAGCGGTGGTGGTGATGT | 118381–118402 | |
US6 R | GCGGATGGGCGATGGAGAAGAG | 120665–120686 | |
US7 F | TGCCCAGAAAGCCAAAAAAG | 120085–120104 | |
US7 R | TCAGCCCGAAAAGCAATAAC | 121767–121786 | |
US8 F | CGCGAGAGGGTTCGAAAAGGGC | 121684–121705 | |
US8 R | CGCCTCGATAGTTTTCGTTGAC | 123569–123590 | |
US9 F | CTGTGCCGTCTGACGGAAAGCA | 123461–123482 | |
US9 R | TATATCTTGTGGTTCTAGTTGTT | 124429–124451 |
px459-gE-sgRNA1/px459 | px459-gE-sgRNA2/px459 | px459-gE-sgRNA1/sgRNA2 | |
---|---|---|---|
BoHV-1 gE/EGFP+ | 0 | 0 | 1 |
Plaque number | 200 | 200 | 200 |
HR efficiency | 0 | 0 | 0.5% |
px459-EGFP-sgRNA1/px459 | px459-EGFP-sgRNA2/px459 | px459-EGFP-sgRNA1/sgRNA2 | |
---|---|---|---|
Edited EGFP | 7 | 3 | 16 |
BoHV-1 gE/EGFP− | 0 | 0 | 6 |
Plaque number | 200 | 200 | 200 |
sgRNA editing efficiency | 3.5% | 1.5% | 8% |
HR efficiency | 0 | 0 | 3% |
px459-EGFP-sgRNA1/px459 | px459-EGFP-sgRNA2/px459 | px459-EGFP-sgRNA1/sgRNA2 | |
---|---|---|---|
Edited EGFP | 11 | 4 | 18 |
BoHV-1 gE/US9/EGFP− | 0 | 0 | 0 |
Plaque number | 200 | 200 | 200 |
sgRNA editing efficiency | 5.5% | 2% | 9% |
HR efficiency | 0 | 0 | 0 |
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Liu, C.-Y.; Jin, M.; Guo, H.; Zhao, H.-Z.; Hou, L.-N.; Yang, Y.; Wen, Y.-J.; Wang, F.-X. Concurrent Gene Insertion, Deletion, and Inversion during the Construction of a Novel Attenuated BoHV-1 Using CRISPR/Cas9 Genome Editing. Vet. Sci. 2022, 9, 166. https://doi.org/10.3390/vetsci9040166
Liu C-Y, Jin M, Guo H, Zhao H-Z, Hou L-N, Yang Y, Wen Y-J, Wang F-X. Concurrent Gene Insertion, Deletion, and Inversion during the Construction of a Novel Attenuated BoHV-1 Using CRISPR/Cas9 Genome Editing. Veterinary Sciences. 2022; 9(4):166. https://doi.org/10.3390/vetsci9040166
Chicago/Turabian StyleLiu, Chun-Yu, Ming Jin, Hao Guo, Hong-Zhe Zhao, Li-Na Hou, Yang Yang, Yong-Jun Wen, and Feng-Xue Wang. 2022. "Concurrent Gene Insertion, Deletion, and Inversion during the Construction of a Novel Attenuated BoHV-1 Using CRISPR/Cas9 Genome Editing" Veterinary Sciences 9, no. 4: 166. https://doi.org/10.3390/vetsci9040166