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 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.
Three vectors are included in the kit: pMTL575555 (stCas91), pMTL675555 (stCas93) and pMTL-RiboCas93 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 [1]. The vector pMTL-RiboCas93, based on pMTL675555, contains a Pgapdh RbxE riboswitch upstream of Cas9 [2].
References
[1] 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.
https://doi.org/10.1021/acssynbio.1c00138
[2] Matthew S. H. Lau, Abubakar Madika, Ying Zhang, and Nigel P. Minton. Parageobacillus thermoglucosidasius Strain Engineering Using a Theophylline Responsive RiboCas for Controlled Gene Expression. ACS Synth Biol. 2024 13 (4), 1237-1245.
https://doi.org/10.1021/acssynbio.3c00735