Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant.

ABSTRACT: 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

SUBMITTER: Adalsteinsson BT 

PROVIDER: S-EPMC8100143 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature