Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
05 05 2021
05 05 2021
Historique:
received:
18
12
2020
accepted:
20
04
2021
entrez:
6
5
2021
pubmed:
7
5
2021
medline:
3
2
2023
Statut:
epublish
Résumé
Thermophilic organisms are extensively studied in industrial biotechnology, for exploration of the limits of life, and in other contexts. Their optimal growth at high temperatures presents a challenge for the development of genetic tools for their genome editing, since genetic markers and selection substrates are often thermolabile. We sought to develop a thermostable CRISPR-Cas9 based system for genome editing of thermophiles. We identified CaldoCas9 and designed an associated guide RNA and showed that the pair have targetable nuclease activity in vitro at temperatures up to 65 °C. We performed a detailed characterization of the protospacer adjacent motif specificity of CaldoCas9, which revealed a preference for 5'-NNNNGNMA. We constructed a plasmid vector for the delivery and use of the CaldoCas9 based genome editing system in the extreme thermophile Thermus thermophilus at 65 °C. Using the vector, we generated gene knock-out mutants of T. thermophilus, targeting genes on the bacterial chromosome and megaplasmid. Mutants were obtained at a frequency of about 90%. We demonstrated that the vector can be cured from mutants for a subsequent round of genome editing. CRISPR-Cas9 based genome editing has not been reported previously in the extreme thermophile T. thermophilus. These results may facilitate development of genome editing tools for other extreme thermophiles and to that end, the vector has been made available via the plasmid repository Addgene.
Identifiants
pubmed: 33953310
doi: 10.1038/s41598-021-89029-2
pii: 10.1038/s41598-021-89029-2
pmc: PMC8100143
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
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