Expanding the CRISPR toolbox for use within Bacillus subtilis

Abstract

DNA editing is a vital tool in the development of biological systems for both research and commercial applications. Novel enabling tools accelerate strain engineering for the study of cellular mechanisms or production of small molecules and proteins. CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) technologies, where small RNA molecules (gRNA) direct Cas (CRISPR-associated) proteins to DNA in a highly accurate, sequence-dependent manner have increased the rate at which DNA modifications can be made.

We developed and expanded the CRISPR toolbox for recombination and deaminase-guided genome editing, protein engineering and transcriptional regulation within the industrial workhorse Bacillus subtilis.

A co-transformational system, consisting of a single plasmid for nuclease and gRNA expression and a linear donor DNA (dDNA) was established for use with both Streptococcus pyogenes Cas9, the most commonly utilised CRISPR nuclease, as well as the potentially highly commercially relevant nuclease, MAD7, also known as Eubacterium rectale Cas12a. Editing efficiencies of ≥83% were observed for both nucleases.

Using our CRISPR-Cas9 genome editing tool, a novel variant of the commercially relevant protein, subtilisin E, was engineered exhibiting an increase in both thermostability and proteolytic efficiency. Two systems for transcriptional down-regulation (CRISPRi) were demonstrated, including the first reported catalytically inactive variant of MAD7. Finally, Cas9 was further modified to incorporate fusions with DNA deaminases allowing the first reported example of CRISPR targeted deaminase base editing within B. subtilis.