Novel and efficient CRISPR/Cas systems for precise genome editing of industrially relevant microorganisms

CRISPR/Cas has revolutionized genome editing and enables the precise genetic modification of various single-celled organisms in biotechnology. However, the commercial use of the well-characterized nucleases is severely hindered due to patent and licensing uncertainties.

To overcome these limitations, we have identified novel Cas nucleases with little or no homology to database Cas proteins in an elaborate metagenomics sequencing analysis of rationally selected environmental samples. Among others, several potential class 2 type V CRISPR systems suitable for genome editing resulted from this metagenome screening of over 3 tera base pairs in sequencing data. In order to evaluate the genome editing activity of our in silico identified sequences, we carried out experiments in E. coli as the classical prokaryotic model organism. We were able to show that our novel Cas nucleases, which are termed G-dase M, can be programmed to efficiently and precisely introduce a genomic DNA double-strand break. Using an additional bacterial non-homologous end joining (NHEJ) system, we then introduced gene-specific INDEL mutations with different G-dase M nucleases. Furthermore, one chosen G-dase M system in combination with the λ/Red recombinase system enabled efficient homology-directed repair (HDR) with provided DNA repair fragments in E. coli. We have thus proven that we were indeed able to identify functional and novel Cas proteins.

To also evaluate the activity of our G-dase M nucleases in a eukaryotic organism, we have adapted one selected system to genetically modify various Saccharomycetes laboratory strains and diploid commercial wine starter cultures in a targeted and efficient manner. This utilized the cell's own ability to repair introduced DNA double-strand breaks by HDR. We have even established a powerful single-plasmid system for a winemaking yeast for which no CRISPR system has been described so far. All the results of our genome editing studies have shown that our proprietary CRISPR systems are valuable tools for the genetic modification of prokaryotic and eukaryotic organisms.