Difference between revisions of "Part:BBa K2017000"

Revision as of 11:14, 21 October 2016

C-split Cas9 + DnaE C-intein

Half of the SpCas9 protein fused to DnaE C-intein. The active full-lenght spCas9 can be recovered by using the other half of the protein, N-split Cas9 + DnaE N-intein. Inteins are used to fuse both parts of the protein, as they interact and splice, leaving the active protein.

Usage and Biology

The protein Cas9 is used in the editing system CRISPR/Cas9. The protein is an endonuclease that cleaves DNA at an specific point, determined by the guide RNA. The guide RNA, which has the structure G-19-NGG, binds the Cas9 and guides it to the DNA strand complementary to the gRNA. Once it recognizes the region to edit, it cuts three nucleotides upstream the recognition sequence, NGG. It is a powerful tool for gene editing.

In this case the Streptococcus pyogenes Cas9 (SpCas9) is the one used in the Valencia UPV iGEM project. The lenght of the Cas9 is 4137bp. We aim to deliver the Cas9 in plants to perform gene editing. As we want to make the editing more efficient, instead of using Agrobacterium tumefaciens as carrier of the Cas9, we use plant viral vectors. However, conventional viral vectors usually are able to carry around 2kb. The SpCas9 is too large. Our solution is to divide the Cas9 in to parts, obtaining a split-Cas9 which can be inserted in viral vector.

To join both parts of the split-Cas9 and reconstitute the functional Cas9 protein, we have used N and C inteins, (BBa_K1362400 and BBa_K1362401, of the 2014 Heidelberg team. These inteins show splicing activity once they join each other. We have added N-intein to the N split-Cas9 (BBa_K2017001) and C-intein to the C split-Cas9, so when they are translated inside the plant cell, the inteins join, splice themselves and let the Cas9 protein complete and fully functional.

References

Truong D, Kühner K, Kühn R, Werfel S, Engelhardt S, Wurst W et al. Development of an intein-mediated split–Cas9 system for gene therapy. Nucleic Acids Res. 2015;43(13):6450-6458.

Vazquez-Vilar M, Bernabé-Orts J, Fernandez-del-Carmen A, Ziarsolo P, Blanca J, Granell A et al. A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 2016;12(1).

Khatodia S, Bhatotia K, Passricha N, Khurana S, Tuteja N. The CRISPR/Cas Genome-Editing Tool: Application in Improvement of Crops. Frontiers in Plant Science. 2016;7.

Li J, Norville J, Aach J, McCormack M, Zhang D, Bush J et al. Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotechnology. 2013;31(8):688-691.

Nekrasov V, Staskawicz B, Weigel D, Jones J, Kamoun S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature Biotechnology. 2013;31(8):691-693.

Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology. 2013;31(8):686-688.

Sequence and Features

Functional Parameters