The Inscripta Blog

November 16, 2021

Expanding the gene editing toolbox for Bacillus subtilis with the MAD7 nuclease

What would microbial biotechnology do without its heroes, E. coli and S. cerevisiae? The utility of these workhorses is undisputable, but other microbes are also gaining traction as desirable hosts for biomolecule and chemical production due to some of the unique properties of these alternative microbes. The challenge with using these more exotic microbes is that efficient genetic engineering tools are not yet available in these microbes. This is why we are excited to see our CRISPR editing MAD7 nuclease expanding the gene editing toolbox for alternative hosts.

A recent review article “The CRISPR toolbox for the gram-positive model bacterium Bacillus subtilis” published last month in Critical Reviews in Biotechnology highlights the progress in developing CRISPR tools in this bacterium. B. subtilis is a model gram-positive organism capable of growing well on inexpensive carbon sources and is one of the bacterial champions used in secreted enzyme production. One of its unique value propositions is that B. subtilis is generally regarded as a safe (GRAS) microbial host, allowing it to be used in food-grade applications, industrial fermentation, and bioremediation. Therefore, conventional genome engineering methods that require the use of antibiotic resistance markers cannot be used in this eco-friendly host.

CRISPR editing offers a way to introduce or edit genes and pathways without the use of antibiotics. CRISPR technology was first adapted in B. subtilis in 2016. Earlier this year, a research team from the University of Edinburgh, UK, published their work on the use of the MAD7 nuclease to edit the B. subtilis 168 strain. They demonstrated gene knock-out efficiency as high as 93% with CRISPR-MAD7, comparable to that of CRISPR-Cas9, and up to 100% editing efficiency for point mutations. They also engineered a catalytically inactive MAD7 (dMAD7) nuclease variant and showed that it was capable of reducing gene expression of single and multiple targets up to 71.3% via CRISPR interference mechanism.

Authors noted several advantages of using the MAD7 nuclease, including T-rich PAM preferences and the smaller size of the nuclease. But the main advantage they cited is the IP-friendly nature of the MAD7 enzyme from Inscripta. The nuclease sequence has been publicly released by Inscripta and it is free for use in academic and industrial R&D applications for most applications, enabling scientists to use CRISPR editing to make discoveries and translate them into products without reach-through royalties. The MAD7 and other MADzyme™ nuclease licenses are also available on commercially reasonable terms for those who are interested in getting access to MAD7 or enzyme variants with specific properties.

To learn more about the MAD7, MADzyme nucleases and licensing, visit our website.