Part:BBa_K3044020
sgRNA/dCas9 system targeting mRFP
The assembled sgRNA-dCas9 system can be used for silencing of mRFP (BBa_B1010) expression.
This dCas9 is a codon optimized version of BBa_K1150000 for expression in E. coli. It forms a complex with the single guide RNA (sgRNA), and together this system can be used for silencing of specific target genes. dCas9 is the inactive form of the endonuclease Cas9. In contrast to Cas9, dCas9 does not cleave the DNA. Instead, it sterically blocks the elongation of transcription as a result of the sgRNA-DNA interaction. This will only repress the expression of the target gene thus avoiding destruction of the gene. [1]
This sgRNA sequence targets residue 325-245 on the template strand in the mRFP sequence and have an efficiency of 67.06. When forming a complex with the dCas9, the sgRNA guides the dCas9 to the appropriate target gene. The sgRNA is designed according to the location of the PAM sequence in the target gene, because the PAM sequence is recognized by and activates the dCas9. The sgRNA consists of 100 nucleotides of which 20 nucleotides base pair with the target DNA sequence and the rest is called a dCas9 handle and interacts with the dCas9 protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 478
Illegal BglII site found at 1552 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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