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Part:BBa_K2924005
guideRNA from long-chain-fatty-acid CoA ligase
long-chain-fatty-acid CoA ligase slr1609 guide RNA of Synechocystis sp.
Usage and Biology
This part contains the long-chain-fatty-acid CoA ligase guide RNA of Synechocystis sp. PCC 6803. It was used for an induced knock-down of slr1609 with a CRISPRi/dCas9-system, which was kindly provided by Yao et al. (2015)4. The long-chain-fatty-acid CoA ligase can be found under the UniProt ID: P73004_SYNY31 and is involved in fatty acid synthesis, degradation, and metabolism2. The gene is positioned in the genome at 487287 - 489377 (2091 bp) bases 2. The guide RNA was obtained by using the CRISPR guide from benchling3. The sgRNA in the gene is located at 201-220 bp in the + strand (Fig. 1). The sequence of the sgRNA is CCATTCCATCCATTGCCTGG, has anOn-Target Score of 72.2 and an Off-Target Score of 50.0.
The long-chain-fatty-acid CoA ligase catalyzes the pre-step reaction for β-oxidation of long-chain fatty acids by ligating coenzyme A to a fatty acid under consumption of a lot of energy in the form of ATP 5. These enzyme is present in all organisms from bacteria to human. Its mechanism is well known and can be divided in several steps.
Step one: One ATP molecule and a long-chain fatty acid enter the active site of the enzyme. The negatively charged oxygen of the fatty acid attacks the ATP and forms a AMP-fatty acid intermediate. Step two: Pyrophosphate leaves the active site. Step three: Coenzyme A enters the active site and forms with the AMP-fatty acid intermediate another one, the AMP-fatty acid-CoA. Step four: At the end, fatty acid-CoA and AMP are released out of the active site (Fig. 2)5.
This enzyme dimerizes and is then able to bind ATP at the C-terminal and fatty acid at the binding tunnel of the N-terminal, which can now interact with each other by different interaction between C- and N-terminal 5.
The short guide RNA (sgRNA) was cloned into a vector containing a neutral site of Synechocystis sp. PCC 6803. That’s a homologous sequence of its genome to ensure a knock-in into the genome (Fig. 3)4.
Due to this knock-in containing a resistance for antibiotic and the sgRNA, we can down-regulate the target enzyme with a CRISPRi/dCas9 - system 4. This system is induced by anhydrotetracycline (aTc), which activates the synthesis of the dCas9, which is then binding to the sgRNA. These complex is able to bind complementary to the targeted enzyme and stops the transcription of it (Fig. 4).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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