2019-11-14| Technology

Ignavibacterium Cas9 Beats Thermostability Record, Functions at 100 Degree Celsius

by Rajaneesh K. Gopinath
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By Rajaneesh K. Gopinath, Ph.D.

Researchers isolate a novel heat resistant Cas9 nuclease from a hyperthermophilic bacterium that grows in a high-temperature environment.

The CRISPR-Cas9 genome-editing tool has evolved rapidly since its discovery and it continues to impact various biological disciplines including genetic engineering. The technique undergoes incessant upgradations and is currently tested for various clinical applications.

Since the discovery of the system, scientists have isolated and tweaked Cas9 nucleases from various mesophilic bacteria with nearly all of them functioning at moderate temperatures. The most predominantly used Streptococcus pyogenes Cas9 (SpyCas9) has a limit of 44°C. The two exceptions are the thermostable Cas9s isolated from Geobacillus stearothermophilus (GeoCas9) and Geobacillus thermodenitrificans T12 (ThermoCas9) which have a limit of 70°C.


A New Thermostable Cas9 in Town

In a collaborative effort, researchers from Standford, MIT, and the Broad Institute have isolated a novel Cas9 nuclease from a hyperthermophilic bacterium called Ignavibacterium (IgnaviCas9). By using microfluidic mini-metagenomic sequencing of a sediment sample from the Lower Geyser Basin of Yellowstone National Park, the group discovered that the bacterial genome contained a full CRISPR array. The temperature of the organism’s habitat itself exceeded 90°C which intrigued the authors to express, purify, and characterize the novel Cas9 protein.

They found that IgnaviCas9 also belongs to type II-C Cas9, a system that employs three Cas proteins with Cas9 playing the major role. However, it is highly divergent from the two previously isolated thermostable Cas9s operating beyond their temperature range. IgnaviCas9 was found to be active even at 100°C.

To prove the significance of their finding, the authors demonstrated that the nuclease can be used in bacterial RNA-seq library preparation by removing unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps. The authors believe that IgnaviCas9 will undoubtedly be an asset in developing new biotechnological techniques and advance the field further. The findings were published in the Proceedings of the National Academy of Sciences Journal.




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