Translational_Unit
RGR-gRNA

Part:BBa_K2538010:Design

Designed by: Mor Pasi   Group: iGEM18_BGU_Israel   (2018-09-03)


Ribozyme flanked gRNA for SadCas9 VP64 targeting synthetic activation promoter pMLPm


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 145
    Illegal NgoMIV site found at 174
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Designed using Geneious to meet registry standards. Part was synthesized by Syntezza and cloned into pSB1C3 using EcoRI and PstI restriction sites.

Source

Sequence of gRNA spacer: Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. Farzadfard F, Perli SD, Lu TK. ACS Synth Biol. 2013 Oct 18;2(10):604-13. doi: 10.1021/sb400081r. Epub 2013 Sep 11. http://www.ncbi.nlm.nih.gov/pubmed/23977949

Sequence of gRNA SaScaffold: In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature. 2015 Apr 1. doi: 10.1038/nature14299. 10.1038/nature14299 PubMed 25830891

Ribozyme-flanked (RGR) design for the gRNA synthesis under RNA polymerase II promoters: Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Gao Y, Zhao Y. J Integr Plant Biol. 2014 Apr;56(4):343-9. doi: 10.1111/jipb.12152. Epub 2014 Mar 6. http://www.ncbi.nlm.nih.gov/pubmed/24373158 Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Mol Cell. 2014 May 22;54(4):698-710. doi: 10.1016/j.molcel.2014.04.022. Epub 2014 May 15. http://www.ncbi.nlm.nih.gov/pubmed/24837679


References

1. Farzadfard, Fahim, Samuel D. Perli, and Timothy K. Lu. "Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas." ACS synthetic biology 2.10 (2013): 604-613.‏

2. Nissim, Lior, et al. "Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells." Molecular cell 54.4 (2014): 698-710.‏

3. Gao, Yangbin, and Yunde Zhao. "Self‐processing of ribozyme‐flanked RNAs into guide RNAs in vitro and in vivo for CRISPR‐mediated genome editing." Journal of integrative plant biology56.4 (2014): 343-349.‏

4. Ran, F. Ann, et al. "In vivo genome editing using Staphylococcus aureus Cas9." Nature 520.7546 (2015): 186.‏

5. Perez-Pinera, Pablo, et al. "RNA-guided gene activation by CRISPR-Cas9–based transcription factors." Nature methods10.10 (2013): 973.‏

6. Kim, Jin Hee, et al. "High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice." PloS one 6.4 (2011): e18556.‏