Open Readings 2021 • P8-15
CONSTRUCTION OF TYPE III-A CRISPR-CAS EFFECTOR COMPLEX FOR TRANSPORTATION INTO THE ZEBRAFISH NUCLEUS
Danas Klimavičius1, Dalia Smalakyte1, Abhishek Pateria2, Matthias Bochtler2, 3, Gintautas Tamulaitis1
1 Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio av. 7, 10257 Vilnius, Lithuania
2 International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland
3 Institute of Biochemistry and Biophysics PAS, Pawinskiego 5a, 02-106 Warsaw, Poland
[email protected]
RNA knockdown is a powerful experimental technique to study gene function and it is commonly mediated through RNA interference. However, some organisms such as zebrafish cannot tolerate the additional load on their interfering RNA machinery [1]. Our group proposed that type III-A CRISPR-Cas effector complex Csm from Streptococcus thermophilus (StCsm) could be used for RNA knockdown as a programmable ribonuclease in this model organism. We demonstrated greater than 50% efficiency of StCsm in RNA knockdown and hence provided a proof of principle of programmable mRNA degradation by StCsm in eukaryotes [2].
Recently it was shown that type I CRISPR-Cas effector complex Cascade, which is of similar size to Csm, could be successfully transported into the nucleus by attaching the nucleus localization sequence (NLS) [3]. Since DNA transcription occurs in the nucleus, synthesized transcripts could be cleaved immediately before transporting them into the cytoplasm. In order to further improve transcript degradation efficiency we decided to deliver StCsm into the nucleus of a cell. In this study we present construction of StCsm variants optimized for transportation into zebrafish nucleus.
[1] Liu W., Wang Y., Sun Y., Wang Y., Wang Y., Chen S., Zhu Z. (2005) Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase. Development Growth and Differentiation, 47(5), p. 323-331
[2] Fricke T., Smalakyte D., Lapinski M., Pateria A., Weige C., Pastor M., Kolano A., Winata C., Siksnys V., Tamulaitis G., Bochtler M. (2020) Targeted RNA knockdown by Type III-A CRISPR-Cas Complex in Zebrafish. The CRISPR Journal, 3(4), p. 299-313
[3] Dolan A., Hou Z., Xiao Y., Gramelspacher M., Heo J., Howden S., Freddolino P., Ke A., Zhang Y. (2019) Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas. Molecular Cell, 74(5), p. 936-950.
