Part:BBa_K4907039
sgRNA (relaxase_pMUT2) sequence
sgRNA(relaxase_pMUT2)
A designed N20 sequence targeting a genetic sequence of relaxase on pMUT2 in E. coli Nissle 1917.
Biology
There are two components in CRISPR system, a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein). The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined 20 nucleotides spacer (called N20 sequence) that decides the target genes to be modified. By changing the N20 sequence, one can change the gene target of the Cas protein. Through the recognition on PAM sequence and the guidance of sgRNA, the Cas proteins eventually bind to the target sequence and display its endonuclease activity (1, 2).
Usage and Design
By harnessing the pEcCas/pEcgRNA system (CRISPR/Cas9 based) (3), we carried out the elimination of pMUT2 in E. coli Nissle 1917. By reviewing the article (4), we get the CRISPR array and modified it to a suitable N20 sequence. Then, it was constructed and assembled to the pEcgRNA backbone which had undergone digestion by Bsa I. After successful construction, BBa_K4907039_pEcgRNA was then transformed into the E. coli Nissle 1917 with pEcCas and the elimination of pMUT2 was carried out.
Characterization
Agarose Gel Electrophoresis
When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (410 bp) can be observed at the position between 250 and 500 bp.
Reference
1. K. Wang, Y. Wang, Basic properties and research methods of a new CRISPR-Cas system: CasX as an example. Microbiology China 48 , 1227-1238 (2021).
2. F. Jiang, J. A. Doudna, CRISPR–Cas9 structures and mechanisms. Annu. Rev. Biophys. 46, 505-529 (2017).
3. Q. Li et al., A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim. Biophys. Sin. 53, 620-627 (2021).
4.A. Kan, I. Gelfat, S. Emani, P. Praveschotinunt, N. S. Joshi, Plasmid vectors for in vivo selection-free use with the probiotic E. coli Nissle 1917. ACS Synth. Biol. 10, 94-106 (2020).
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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