Part:BBa_K3904403:Design

adhE-specific sgRNA

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]

Design Notes

gRNA scaffold is universal for sgRNA used in the composite with Cas9 nuclease. However, gRNA should be carefully selected before each experiment. Things to consider during the search of gRNA:

1. Off-target rate. It is very important to consider whether chosen sgRNA will not target other genomic sites.
2. GC-content. It is estimated that lower GC content might cause Cas9 difusion from the DNA site and lead to the low cleavage efficiency. However, GCC motif also should be avoided.(1)
3. T-rich sequences 3′ end of the targeting sequence.(1)
4. Cleavage efficiency. This could be calculated with some algorithms. In this approach we have used [http://crispor.tefor.net/ CRISPOR] (2).

Source

gRNA has been acquired from E.coli Nissle 1917 genome (NCBI accession number: CP022686) and gRNA scaffold has been acquired from pTargetF plasmid.

References

1. Graf, R., Li, X., Chu, V. T., & Rajewsky, K. (2019). sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing. Cell reports, 26(5), 1098–1103.e3.
2. Haeussler, M., Schönig, K., Eckert, H., Eschstruth, A., Mianné, J., Renaud, J. B., ... & Concordet, J. P. (2016). Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome biology, 17(1), 1-12.