RNA

Part:BBa_K4907039

Designed by: Chern Fang Cheng   Group: iGEM23_XMU-China   (2023-09-14)


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.

Fig. 1 The map of pMUT2 showing the target sequence of sgRNA.


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.

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907039_pEcgRNA.


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None