S. Schmidt, F. Yu, P. Blainey
Oct 28, 2019
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0
Influential Citations
16
Citations
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abstract
Significance Cas9 proteins have revolutionized biotechnology by enabling flexible and facile targeted cleavage of nucleic acids. Nearly all of these proteins are active only at moderate, near-physiological temperatures. Through mini-metagenomic sequencing of hot spring samples from Yellowstone National Park, we discovered and characterized a novel hyperthermophilic Cas9 protein from an unculturable Ignavibacterium. This Cas9 protein, IgnaviCas9, expands the temperature range at which targeted nucleic acid cleavage is possible, thus speeding the development of new biotechnological techniques. We demonstrated one such application by using IgnaviCas9 to deplete undesired amplicons during the amplification step of library preparation in sequencing workflows. This Cas9 protein underscores the exciting applications that can be made possible by exploring nature’s diversity. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (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 demonstrate that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.