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 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.

A Hyperthermoactive-Cas9 Editing Tool Reveals the Role of a Unique Arsenite Methyltransferase in the Arsenic Resistance System of Thermus thermophilus HB27

“…For this purpose, a marker-free homologous recombination and ThermoCas9-based counterselection genome-editing tool was developed which was highly efficient and active at temperatures up to 65°C. Our tool equals the highest reported temperature for a Cas9-based editing tool to date ( 51 , 52 ). The characterization of the T. thermophilus ΔTtarsM strain confirmed its expected higher sensitivity to arsenite, but not arsenate, than the wild-type strain.…”

Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering

“…The selection of prokaryotic cells that have undergone mutations in genome editing experiments often requires laborious experimentation and Cas9 nucleases have been harnessed to simplify this task [26,59,67,68]. Here we have applied EHCas9 to positively select E. coli cells that had experienced deletion of a chromosomal target after recombinase-mediated homologous recombination, without requiring the introduction of sequences such as selection markers in the host genome.…”

Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes

“…Genome edition in T.th. with CaldoCas9 was adapted from Adalsteinsson et al (2021) . A total of 30 nt long ssDNA oligonucleotides containing spacers were designed with 4 nt long overhangs at 5′-end, complementary to Bpi I (ThermoFisher Scientific; ref: ER1011) digested pTTCC.…”