Part:BBa_K3044026
sgRNA/Cas9 system targeting mRFP
The assembled sgRNA-Cas9 system can be used for knockout of mRFP (BBa_B1010) gene expression.
This Cas9 is an active version of (dCas9) with the A10D and A840H mutations to activate the catalytic domains RuvC and HNH. Cas9 has endonuclease activity and induces a double stranded break in the target DNA sequence. The protein forms a complex with the single guide RNA (sgRNA), and together this system can be used for knockout of specific target genes.
This sgRNA sequence targets residue 490-510 on the template strand in the mRFP sequence and have an efficiency of 72.68. When forming a complex with the Cas9, the sgRNA guides the Cas9 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 Cas9. The sgRNA consists of 100 nucleotides of which 20 nucleotides base pair with the target DNA sequence and the rest is called a Cas9 handle and interacts with the Cas9 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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