Baculovirus Variant Detection from Transient CRISPR-Cas9-Mediated Disruption of gp64 at Different Gene Locations
Abstract
1. Introduction
2. Results
2.1. Effect of gp64 Gene Disruption on Foreign Protein and Progeny Virus Production
2.2. Confirmation of CRISPR-Cas9-Mediated Gene Editing of gp64
2.3. Variant Generation or Conservation over Passages
2.4. Evaluation of Mutations Outside the Targeted gp64 Gene
3. Discussion
3.1. CRISPR-Cas9-Mediated Targeted Disruption of gp64 During Virus Propagation
3.2. Are CRISPR-Cas9 Off-Targets Observed in Our System?
3.3. Variant Conservation or Random Mutations upon Virus Propagation in Cell Culture
4. Materials and Methods
4.1. Cell Line and Maintenance
4.2. Plasmid Design and Construction
4.3. Baculovirus Amplification and Quantification
4.4. Transfection-Infection Assay (T-I Assay)
4.5. Flow Cytometry Analysis of GFP upon gp64 Gene Disruption
4.6. Total Baculovirus Quantification via Flow Cytometry
4.7. Tiled-Amplicon Sequencing Assay for rBEV Genomes
4.8. Bioinformatics Pipeline for Minor Species
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AcMNPV | Autographa californica multiple nucleopolyhedrovirus |
au | arbitrary units |
BEVS | baculovirus expression vector system |
DIPs | defective interfering particles |
EPDA | end-point dilution assay |
GFP | green fluorescent protein |
HDR | homology-directed repair |
hpi | hours post-infection |
hpt | hours post-transfection |
hrs | homologous repeat regions |
IDT | integrated DNA technologies |
IVT | infectious virus titer |
MOI | multiplicity of infection |
NHEJ | non-homologous end joining |
NGS | next-generation sequencing |
ORF | open reading frame |
ori | origin of replication |
PAM | protospacer adjacent motif |
PBS | phosphate-buffered saline |
pfu/mL | plaque-forming units per mL |
rBEV | recombinant baculovirus expression vector |
SFM | serum-free media |
sgRNA | single-guide RNA |
T-I assay | transfection-infection assay |
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Target 1 | Estimated Position (bp) 2 | Observed Position (bp) [Mutation] (Frequency) 3 | |
---|---|---|---|
Cell Pellet | Supernatant | ||
gp64+131 | 107,222–107,227 | 107,222 [−C] (6.62%) | Not observed |
gp64−160 | 107,199–107,204 | 107,201 [−CCA] (6.30%) 107,202 [−CA] (8.47%) | 107,199 [−CTCCA] (3.05%) 107,200 [−TCCA] (3.48%) 107,201 [−CCA] (5.44%) 107,202 [−CA] (6.34%) 107,204 [−C] (1.43%) |
gp64+278 | 107,075–107,080 | Not observed | Not observed |
gp64+378 | 106,975–106,980 | 106,975 [−T] (4.51%) | 106,975 [−T] (4.04%) |
gp64+418 | 106,935–106,940 | 106,935 [−CA] (4.24%) | 106,935 [−CA] (2.17%) |
gp64+131/384 | 107,222–107,227 106,969–106,974 | Not observed | Not observed |
Mutation Region | Mutation Type | Virus Stock Consensus/ Virus Stock Variant/ T-I Assay Variant 1 | Mutation Position |
---|---|---|---|
AcOrf-84 promoter | SNPs | G/T/T | 71,443 |
fgf 3′ UTR | SNPs | A/G/G | 27,502 |
fgf 3′ UTR | SNPs | G/A/A | 27,505 |
fgf 3′ UTR | SNPs | G/A/A | 27,506 |
fgf 3′ UTR | SNPs | G/A/A | 27,509 |
AcOrf-603 | SNPs | C/T/T | 3960 |
AcOrf-1629 | SNPs | G/A/A | 6375 |
lef10 | SNPs | G/A/A | 45,761 |
AcOrf-91 | SNPs | T/A/A | 78,627 |
AcOrf-91 | SNPs | A/T/T | 78,666 |
AcOrf-1629 | Insertion | -/GATC/GATC | 7304 |
AcOrf-51 | Insertion | -/A/A | 44,073 |
egt | Deletion | 2 CTAGAGA/-/- | 12,427 |
AcOrf-91 | Deletion | TAT/-/- | 78,926 |
Gene | Location | Spacer Sequence (5′-3′) | PAM | Strand |
---|---|---|---|---|
Scrambled control | N/A | CACCTTGAAGCGCATGAACT | N/A | N/A |
gp64 | +131 | GGAAACGCTGCAAAAGGACG | TGG | Antisense |
gp64 | −160 | GTTGTAGTCCGTCTCCACGA | TGG | Sense |
gp64 | +278 | AACGCTGAATGTGGGCAAAG | AGG | Antisense |
gp64 | +378 | GACTGTTTTCGCGACAACGA | GGG | Antisense |
gp64 | +418 | AAGGCAAAGAGTTGGTGAAG | CGG | Antisense |
gp64 | +131/ 384 | GGAAACGCTGCAAAAGGACG/ TTTCGCGACAACGAGGGCCG | TGG/ CGG | Antisense |
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Chakraborty, M.; Nielsen, L.; Nash, D.; Bruder, M.R.; Nissimov, J.I.; Charles, T.C.; Aucoin, M.G. Baculovirus Variant Detection from Transient CRISPR-Cas9-Mediated Disruption of gp64 at Different Gene Locations. Int. J. Mol. Sci. 2025, 26, 5805. https://doi.org/10.3390/ijms26125805
Chakraborty M, Nielsen L, Nash D, Bruder MR, Nissimov JI, Charles TC, Aucoin MG. Baculovirus Variant Detection from Transient CRISPR-Cas9-Mediated Disruption of gp64 at Different Gene Locations. International Journal of Molecular Sciences. 2025; 26(12):5805. https://doi.org/10.3390/ijms26125805
Chicago/Turabian StyleChakraborty, Madhuja, Lisa Nielsen, Delaney Nash, Mark R. Bruder, Jozef I. Nissimov, Trevor C. Charles, and Marc G. Aucoin. 2025. "Baculovirus Variant Detection from Transient CRISPR-Cas9-Mediated Disruption of gp64 at Different Gene Locations" International Journal of Molecular Sciences 26, no. 12: 5805. https://doi.org/10.3390/ijms26125805
APA StyleChakraborty, M., Nielsen, L., Nash, D., Bruder, M. R., Nissimov, J. I., Charles, T. C., & Aucoin, M. G. (2025). Baculovirus Variant Detection from Transient CRISPR-Cas9-Mediated Disruption of gp64 at Different Gene Locations. International Journal of Molecular Sciences, 26(12), 5805. https://doi.org/10.3390/ijms26125805