Nucleic Acid Cleavage with a Hyperthermophilic Cas9 from an Unculturable Ignavibacterium

CRISPR-Cas9 systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro , which enables DNA cleavage beyond the 44 °C limit of Streptococcus pyogenes Cas9 (SpyCas9) and the 70 °C limit of both Geobacillus stearothermophilus Cas9 (GeoCas9) and Geobacillus thermodenitrificans T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrated that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA (rRNA) without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. Taken together, IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.