Part:BBa_K4687035:Design
MAD7+recE/T+pJYS1+PlacM:MADE/TJlacM-g4
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Design Notes
Traditional gene editing methods are not very efficient in Corynebacterium glutamicum. In order to improve the efficiency of gene editing in Corynebacterium glutamicum, we tried to construct a new system that can be expressed in Corynebacterium glutamicum that can perform gene editing efficiently.Whereas CRISPR-MAD7 nuclease has been described to target a wider range of PAM sequences, 5'-YTTN-3' , and exhibit high gene editing activity in microbial systems with small molecular weights, MAD7 can be used for a variety of gene editing. It has been reported that cas9 expression may retard the growth of Corynebacterium glutamicum due to toxicity of the gene product.CRISPR-MAD7 showed low toxicity in Corynebacterium glutamicum cells. Compared with the CRISPR-Cas9 system, CRISPR-MAD7 has a shorter crRNA length and a simpler secondary structure, which makes it more economical and convenient in designing, synthesizing, and transcribing crRNAs. After the optimization of CRISPR-MAD7 system in the experimental process, it can make its gene editing efficiency in Corynebacterium glutamicum reach 100% which is unattainable by other systems.Therefore, we hope to improve the gene editing efficiency in Corynebacterium glutamicum by optimizing the CRISPR-MAD7 system. For our subsequent knockdown of genes in Corynebacterium glutamicum.
Source
CRISPR-MAD7 nuclease was identified in Eubacterium rectale. RecE/T is derived from Bacterial,Archaeal and Plant Plastid Product.The PlacM promoter was synthesized artificially based on the Bacillus subtilis sacB gene.The vector skeleton pJYS1 was derived from the constitutive expression of FnCpf1 and recT in the C.glutamitum.The gRNAs are mainly designed based on the gene sequence of the PAM site. The sequence design mainly relies on the neighboring motifs located in the sequence of the pre-interval region of the PAM sequence (Protospacer Adjacent Motif).