Part:BBa_K4887002

sgRNA (IbGBSSI)

It was the single-guide RNA designed targeting for the gene IbGBSSI (BBa_K4887001).

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]

Results

(1) GBSSI-sgRNA design

The selected sgRNA is located 7 bp downstream of the start codon, with a total sequence length of 27 bp (Fig. 1). sgRNA oligos were obtained by annealing with the designed primers following:

• Oligo 1 (GBSSI-sgRNA1): 5’-GTGGGGTTGGGTCAATTAGCCCTGAGGAGC-3'
• Oligo 2 (GBSSI-sgRNA2): 5’-AACTGGTGGACTTGGAGATGTTCTTGGAGG-3’

Fig. 1 The target region of gene IbGBSSI and the location of the gRNA. Exons are shown as square frames and surrounding introns appear as lines. sgRNA and PAM are highlighted in yellow and green, respectively.

(2) Construction of the backbone vector

The sgRNA oligos were ligated with digestion products of plasmid psgR-Cas9-At using T4 ligase, and the backbone vector of sgRNA (IbGBSSI) was obtained: psgR-Cas9- sgRNA(IbGBSSI). To confirm the correctness of the vector, 1/2 of the DNA was then transform into bacteria and incubate in LB for 45 min, and was plated in LB containing Ampicillin (Fig. 2). The obtained clones were validated by performing PCR detection for the sgRNA sequence with primers M13F/oligo2 (The sequence of M11F was: 5’-TGTAAAACGA CGGCCAGT-3’). The gel electrophoresis results (Fig. 3) showed that the gene band were approximately 100bp, as expected, indicating that backbone vector of sgRNA (IbGBSSI) was been constructed successfully.

Fig. 2 E. Coli transformed with backbone vector of sgRNA (IbGBSSI)

Fig. 3 PCR product of sgRNA (IbGBSSI) in E. Coli transformed with backbone vector of sgRNA (IbGBSSI)