Part:BBa_K4943058
Riboswitch with Cas9 nuclease (codon optimized)
This genetic toolbox allows inducible control of Cas9 nuclease expression for efficient genome editing in Clostridium species. It utilizes a synthetic riboswitch that regulates translation initiation in response to theophylline. The system consists of a promoter (Pfdx+) coupled to a theophylline riboswitch variant followed by the codon optimized Streptococcus pyogenes cas9 gene in our dual plasmid CRISPR system. The documented riboswitch gave the highest dynamic range of Cas9 expression in C.butyricum. To perform genome editing, a single plasmid is constructed containing the riboswitch-cas9 module.The second plasmid with the sgRNA expression cassette and repair template flanked by homology arms. Adding theophylline triggers Cas9 production for creating precise gene knockouts or targeted insertions. The riboswitch-cas9 toolbox enables rapid and efficient modification of clostridial chromosomes without leaving behind antibiotic markers. It has been validated for editing different genomic targets in C. sporogenes, C. pasteurianum and we have validated and optimized this sequence to C butyricum. To implement this system, the cas9 gene and desired riboswitch variant need to be cloned behind a functional clostridial promoter like Pfdx+. The sgRNA/repair template section can be exchanged for different gene targets. Adding theophylline at 2-5 mM during gene editing enhances Cas9 induction. This provides a flexible and optimized riboswitch-Cas9 editing method for Clostridium species, facilitating precise genome modification for metabolic engineering and synthetic biology applications.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 134
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 134
Illegal NotI site found at 5
Illegal NotI site found at 139 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 110
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 134
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 134
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 82
Illegal BsaI site found at 276
Illegal BsaI site found at 337
Illegal BsaI site found at 398
Illegal BsaI site found at 461
Illegal BsaI site found at 520
Illegal BsaI site found at 580
| None |