CRISPR – Cas9
CRISPR-Cas9 Genome-editing Vectors
The CRISPR/Cas9 nuclease of Streptococcus pyogenes has successfully been exploited for prokaryotic genome editing in several different organisms. This CRISPR/Cas9 technology achieves this by identifying and selectively cutting targeted DNA. By first using recombination-based replacement of a target sequence with the desired mutant allele, the progenitor cell population can then subsequently be eliminated through RNA-guided, Cas9-mediated cleavage of the parental allele. However, spCas9 is inactive at temperatures above 42 °C, making its use in P. thermoglucosidasius and other thermophiles problematical. To circumvent this issue, we have developed two Parageobacillus/E.coli shuttle vector CRISPR/Cas9 systems, using the well-characterized CRISPR/Cas9 systems of Streptococcus thermophilus stCas91 and stCas93, to generate gene deletions in P. thermoglucosidasius.
Two vectors are included in the kit (order here), pMTL575555 (stCas91) and pMTL675555 (stCas93), which in addition to Cas9 carry the ColEI and pUB110 origins of replication, a thermostable kanamycin resistance gene, and a locus encoding the specific sgRNA .
 Matthew S. H. Lau, Lili Sheng, Ying Zhang, and Nigel P. Minton. Development of a Suite of Tools for Genome Editing in Parageobacillus thermoglucosidasius and Their Use to Identify the Potential of a Native Plasmid in the Generation of Stable Engineered Strains. ACS Synth Biol. 2021 10 (7), 1739-1749.