Repurposing endogenous type II CRISPR-Cas9 system for genome editing in Streptococcus thermophilus.
CRIPSR-Cas9
CRISPR1
CRISPR3
S. thermophilus
endogenous
genome editing
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
23 Nov 2023
23 Nov 2023
Historique:
revised:
21
10
2023
received:
08
06
2023
accepted:
24
10
2023
medline:
23
11
2023
pubmed:
23
11
2023
entrez:
23
11
2023
Statut:
aheadofprint
Résumé
Streptococcus thermophilus has been extensively used in industrial milk fermentation. However, lack of efficient genetic manipulation approaches greatly hampered the industrial application of this species. Here, we repurposed the endogenous CRISPR1 and CRISPR3 systems, both belong to type II-A CRISPR-Cas9, by delivering a self-targeting CRISPR array with DNA repair template into S. thermophilus LMD-9. We achieved 785-bp deletion in lacZ gene by repurposing CRISPR1 and CRISPR3 systems with efficiencies of 35% and 59%, respectively, when 1-kb DNA repair template was provided. While providing with 1.5-kb repair template, the editing efficiency for deletion in lacZ gene reached 90% using CRISPR3 systems. Diverse editing outcomes encompassing a stop code insertion and single nucleotide variation within lacZ, as well as a 234-bp DNA fragment insertion upstream of ster_0903, were generated with high efficiencies of 75%-100% using the CRISPR3 system. Harnessing the customized endogenous CRISPR3 system to target six genes of eps gene cluster, we obtained six single-gene knockout mutants with efficiencies of 29%-80%, and proved that the epsA, epsE, and epsG were the key genes affecting exopolysaccharides biosynthesis in S. thermophilus LMD-9. Altogether, repurposing the native type II-A CRISPR-Cas9 can be served as a toolkit for precise genome engineering in S. thermophilus for biotechnological applications.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : National Key Research and Development Program of China
ID : 2021YFC2100500
Informations de copyright
© 2023 Wiley Periodicals LLC.
Références
Cui, Y., Jiang, X., Hao, M., Qu, X., & Hu, T. (2017). New advances in exopolysaccharides production of Streptococcus thermophilus. Archives of Microbiology, 199(6), 799-809. https://doi.org/10.1007/s00203-017-1366-1
Fontaine, L., Dandoy, D., Boutry, C., Delplace, B., de Frahan, M. H., Fremaux, C., Horvath, P., Boyaval, P., & Hols, P. (2010). Development of a versatile procedure based on natural transformation for marker-free targeted genetic modification in Streptococcus thermophilus. Applied and Environmental Microbiology, 76(23), 7870-7877. https://doi.org/10.1128/AEM.01671-10
Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, 3rd, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343-345. https://doi.org/10.1038/nmeth.1318
Hao, M., Cui, Y., & Qu, X. (2018). Analysis of CRISPR-Cas system in Streptococcus thermophilus and its application. Frontiers in Microbiology, 9, 257. https://doi.org/10.3389/fmicb.2018.00257
Hidalgo-Cantabrana, C., Goh, Y. J., Pan, M., Sanozky-Dawes, R., & Barrangou, R. (2019). Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus. Proceedings of the National Academy of Sciences, 116, 15774-15783.
Junjua, M., Galia, W., Gaci, N., Uriot, O., Genay, M., Bachmann, H., Kleerebezem, M., Dary, A., & Roussel, Y. (2014). Development of the recombinase-based in vivo expression technology in Streptococcus thermophilus and validation using the lactose operon promoter. Journal of Applied Microbiology, 116(3), 620-631. https://doi.org/10.1111/jam.12376
Kong, L., Song, X., Xia, Y., Ai, L., & Xiong, Z. (2022). Construction of a CRISPR/nCas9-assisted genome editing system for exopolysaccharide biosynthesis in Streptococcus thermophilus. Food Research International, 158, 111550. https://doi.org/10.1016/j.foodres.2022.111550
Kong, L., Xiong, Z., Song, X., Xia, Y., & Ai, L. (2022). CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus. Journal of Dairy Science, 105(8), 6499-6512. https://doi.org/10.3168/jds.2021-21409
Kong, L. H., Xiong, Z. Q., Song, X., Xia, Y. J., Zhang, N., & Ai, L. Z. (2019). Characterization of a panel of strong constitutive promoters from Streptococcus thermophilus for fine-tuning gene expression. ACS Synthetic Biology, 8(6), 1469-1472. https://doi.org/10.1021/acssynbio.9b00045
Lecomte, X., Gagnaire, V., Lortal, S., Dary, A., & Genay, M. (2016). Streptococcus thermophilus, an emerging and promising tool for heterologous expression: Advantages and future trends. Food Microbiology, 53(Pt A), 2-9. https://doi.org/10.1016/j.fm.2015.05.003
Liu, L., Yang, D., Zhang, Z., Liu, T., Hu, G., He, M., Zhao, S., & Peng, N. (2021). High-efficiency genome editing based on endogenous CRISPR-Cas system enhances cell growth and lactic acid production in Pediococcus acidilactici. Applied and Environmental Microbiology, 87(20), e0094821. https://doi.org/10.1128/AEM.00948-21
Magadán, A. H., Dupuis, M. È., Villion, M., & Moineau, S. (2012). Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system. PLoS One, 7(7), e40913. https://doi.org/10.1371/journal.pone.0040913
Markakiou, S., Gaspar, P., Johansen, E., Zeidan, A. A., & Neves, A. R. (2020). Harnessing the metabolic potential of Streptococcus thermophilus for new biotechnological applications. Current Opinion in Biotechnology, 61, 142-152. https://doi.org/10.1016/j.copbio.2019.12.019
Rothstein, S. M., Sen, S., & Mansell, T. J. (2020). Towards high-throughput genome engineering in lactic acid bacteria. Current Opinion in Biotechnology, 61, 181-188. https://doi.org/10.1016/j.copbio.2019.12.015
Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P., & Siksnys, V. (2011). The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Research, 39(21), 9275-9282. https://doi.org/10.1093/nar/gkr606
Selle, K., Klaenhammer, T. R., & Barrangou, R. (2015). CRISPR-based screening of genomic island excision events in bacteria. Proceedings of the National Academy of Sciences, 112, 8076-8081.
Wa, Y., Chanyi, R. M., Nguyen, H. T. H., Gu, R., Day, L., & Altermannd, E. (2022). Extracellular polysaccharide extraction from Streptococcus thermophilus in fermented milk. Microbiology Spectrum, 10(2), 2280-2221.
Zhou, D., Jiang, Z., Pang, Q., Zhu, Y., Wang, Q., & Qi, Q. (2019). CRISPR/Cas9-assisted seamless genome editing in Lactobacillus plantarum and its application in N-acetylglucosamine production. Applied and Environmental Microbiology, 85, e01367-01319. https://doi.org/10.1128/AEM.01367-19.
Zhou, X., Wang, X., Luo, H., Wang, Y., Wang, Y., Tu, T., Qin, X., Su, X., Bai, Y., Yao, B., Huang, H., & Zhang, J. (2021). Exploiting heterologous and endogenous CRISPR-Cas systems for genome editing in the probiotic Clostridium butyricum. Biotechnology and Bioengineering, 118(7), 2448-2459. https://doi.org/10.1002/bit.27753