Abstract
Objectives
To set up an efficient gene editing system in Bacillus thuringiensis (Bt) using CRISPR-Cas9 by demonstrating deletion of chromosomal and plasmid genes.
Results
CRISPR-Cas9 from Streptococcus pyogenes was found to function in Bt cells, resulting in DNA cleavage that is lethal to the cells. The system was assessed for its ability to mediate gene editing by knock-out of the protease genes nprA (neutral protease A) and aprA (alkaline protease A). Gene editing was not detected when the Bacillus-derived pBCX was used to carry CRISPR-Cas9 elements and a DNA repair template. When the Cas9 promoter was replaced with the sporulation-specific promoter cyt2A, a Bt ∆nprA clone was obtained, but this plasmid construct did not give reproducible results. Bt ∆nprA ∆aprA and Bt ∆aprA deletion mutants were finally generated when the Lactobacillus plantarum-derived plasmid pLPPR9 was used, likely due to its lower copy number reducing Cas9 toxicity. Only three to four clones each needed to be screened to identify the desired gene-modified mutants. Conversely, efficient editing of the plasmid vip3A gene required the use of pBCX and longer homology sequences for the repair template.
Conclusions
Capitalizing on the differential impact of plasmid copy number and homology arm length, we devised distinct yet simple and efficient approaches to chromosomal and plasmid gene deletion for Bt that condense the screening process, minimize screening, and facilitate multiple consecutive gene editing steps.
Similar content being viewed by others
References
Bowater R, Doherty AJ (2006) Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining. PLoS Genet 2(2):e8. https://doi.org/10.1371/journal.pgen.0020008
Chakroun M, Banyuls N, Bel Y, Escriche B, Ferré J (2016) Bacterial vegetative insecticidal proteins (Vip) from entomopathogenic bacteria. Microbiol Mol Biol Rev 80(2):329–350. https://doi.org/10.1128/MMBR.00060-15
Cui L, Bikard D (2016) Consequences of Cas9 cleavage in the chromosome of Escherichia coli. Nucleic Acids Res 44(9):4243–4251. https://doi.org/10.1093/nar/gkw223
Doruk T, Gedik ST (2013) An efficient gene deletion system for Bacillus thuringiensis. Biologia 68(3):358–364. https://doi.org/10.2478/s11756-013-0184-4
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3):233–239. https://doi.org/10.1038/nbt.2508
Karlskås IL, Maudal K, Axelsson L, Rud I, Eijsink VG, Mathiesen G (2014) Heterologous protein secretion in Lactobacilli with modified pSIP vectors. PLoS ONE 9(3):e91125. https://doi.org/10.1371/journal.pone.0091125
Klimowicz AK, Benson TA, Handelsman J (2010) A quadruple-enterotoxin-deficient mutant of Bacillus thuringiensis remains insecticidal. Microbiology 156(Pt 12):3575–3583. https://doi.org/10.1099/mic.0.039925-0
Lertcanawanichakul M, Wiwat C (2000) Improved shuttle vector for expression of chitinase gene in Bacillus thuringiensis. Lett Appl Microbiol 31(2):123–128
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Mesrati LA, Tounsi S, Jaoua S (2005) Characterization of a novel vip3-type gene from Bacillus thuringiensis and evidence of its presence on a large plasmid. FEMS Microbiol Lett 244(2):353–358. https://doi.org/10.1016/j.femsle.2005.02.007
Monod M, Denoya C, Dubnau D (1986) Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis. J Bacteriol 167(1):138–147
Peng D, Luo Y, Guo S, Zeng H, Ju S, Yu Z et al (2009) Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis. J Appl Microbiol 106(6):1849–1858. https://doi.org/10.1111/j.1365-2672.2009.04151.x
Promchai R, Promdonkoy B, Tanapongpipat S, Visessanguan W, Eurwilaichitr L, Luxananil P (2016) A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis. J Biotechnol 222:86–93. https://doi.org/10.1016/j.jbiotec.2016.02.019
Reyaz AL, Balakrishnan N, Udayasuriyan V (2019) Genome sequencing of Bacillus thuringiensis isolate T414 toxic to pink bollworm (Pectinophora gossypiella Saunders) and its insecticidal genes. Microb Pathog 134:103553. https://doi.org/10.1016/j.micpath.2019.103553
Selle K, Barrangou R (2015) Harnessing CRISPR-Cas systems for bacterial genome editing. Trends Microbiol 23(4):225–232. https://doi.org/10.1016/j.tim.2015.01.008
Soonsanga S, Rungrod A, Audtho M, Promdonkoy B (2019) Tyrosine-776 of Vip3Aa64 from Bacillus thuringiensis is important for retained larvicidal activity during high-temperature storage. Curr Microbiol 76(1):15–21. https://doi.org/10.1007/s00284-018-1578-x
Tan Y, Donovan WP (2001) Deletion of aprA and nprA genes for alkaline protease A and neutral protease A from Bacillus thuringiensis: effect on insecticidal crystal proteins. J Biotechnol 84(1):67–72
Tan TT, Zhang XD, Miao Z, Yu Y, Du SL, Hou XY et al (2019) A single point mutation in hmgA leads to melanin accumulation in Bacillus thuringiensis BMB181. Enzyme Microb Technol 120:91–97. https://doi.org/10.1016/j.enzmictec.2018.10.007
Wang Y, Zhang ZT, Seo SO, Choi K, Lu T, Jin YS et al (2015) Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. J Biotechnol 200:1–5. https://doi.org/10.1016/j.jbiotec.2015.02.005
Acknowledgements
We would like to thank Dr. Samaporn Teeravechyan for editing the paper. This work was supported by the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency (Grant no. P-18–51565).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
All authors declare that they have no conflict of interest.
Research involving human and animal rights
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Soonsanga, S., Luxananil, P. & Promdonkoy, B. Modulation of Cas9 level for efficient CRISPR-Cas9-mediated chromosomal and plasmid gene deletion in Bacillus thuringiensis. Biotechnol Lett 42, 625–632 (2020). https://doi.org/10.1007/s10529-020-02809-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-020-02809-0