Method of inducible knockdown of essential genes in osc cell culture of Drosophila melanogaster

Capa

Citar

Texto integral

Acesso aberto Acesso aberto
Acesso é fechado Acesso está concedido
Acesso é fechado Somente assinantes

Resumo

In the paper, we propose an RNA interference-based method of inducible knockdown of genes essential for cell viability. The method arranges a genetic cassette in which an inducible metallothionein promoter controls the expression of siRNA precursor. The cassette is inserted into the genomic pre-integrated transgene by CRIPSR-Cas9. The expression of siRNA precursor and following silencing of the gene of interest is activated by the supplementation of the medium with copper ions. This technique with the production of endogenous siRNAs allows the gene knockdown in cell cultures that are refractory to conventional transfection strategies of exogenous siRNA. The efficiency of the developed method was demonstrated in the cell culture of Drosophila ovarian somatic cells for two genes that are essential for oogenesis: Cul3, encoding a component of the multiprotein ubiquitin-ligase complex with versatile functions in proteostasis, and cut, encoding a transcription factor regulating the differentiation of the ovarian somatic cells.

Texto integral

Acesso é fechado

Sobre autores

S. Marfina

National Research Center “Kurchatov Institute”; Mendeleev University of Chemical Technology

Email: n.akulenko11@gmail.com
Rússia, Moscow, 123182; Moscow, 125047

E. Mikhaleva

National Research Center “Kurchatov Institute”

Email: n.akulenko11@gmail.com
Rússia, Moscow, 123182

N. Akulenko

National Research Center “Kurchatov Institute”

Autor responsável pela correspondência
Email: n.akulenko11@gmail.com
Rússia, Moscow, 123182

S. Ryazansky

National Research Center “Kurchatov Institute”

Email: s.ryazansky@gmail.com
Rússia, Moscow, 123182

Bibliografia

  1. Cong L., Ran F.A., Cox D., Lin S., Barretto R., Habib N., Hsu P.D., Wu X., Jiang W., Marraffini L.A., Zhang F. (2013) Multiplex genome engineering using CRISPR/Cas Systems. Science. 339, 819–823.
  2. Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., Norville J.E., Church G.M. (2013) RNA-guided human genome engineering via Cas9. Science. 339, 823–826.
  3. Mohr S., Smith J., Shamu C., Neumüller R., Perrimon N. (2014) RNAi screening comes of age: improved techniques and complementary approaches. Nat. Rev. Mol. Cell. Biol. 15, 591–600.
  4. Ni J.-Q., Zhou R., Czech B., Liu L.P., Holderbaum L., Yang-Zhou D., Shim H.S., Tao R., Handler D., Karpowicz P., Binari R., Booker M., Brennecke J., Perkins L.A., Hannon G.J., Perrimon N. (2011) A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat. Methods. 8, 405–407.
  5. Gilbert L.A., Larson M.H., Morsut L., Liu Z., Brar G.A., Torres S.E., Stern-Ginossar N., Brandman O., Whitehead E.H., Doudna J.A., Lim W.A., Weissman J.S., Qi L.S. (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 154, 442–451.
  6. Xu X., Qi L.S. (2019) A CRISPR–dCas toolbox for genetic engineering and synthetic biology. J. Mol. Biol. 431, 34–47.
  7. Kordyś M., Sen R., Warkocki Z. (2022) Applications of the versatile CRISPR-Cas13 RNA targeting system. WIREs RNA. 13, e1694.
  8. Scharf I., Bierbaumer L., Huber H., Wittmann P., Haider C., Pirker C., Berger W., Mikulits W. (2018) Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells. Oncol. Lett. 15, 2441–2450.
  9. Boettcher M., McManus M.T. (2015) Choosing the right tool for the job: RNAi, TALEN, or CRISPR. Mol. Cell. 58, 575–585.
  10. Housden B.E., Muhar M., Gemberling M., Gersbach C.A., Stainier D.Y.R., Seydoux G., Mohr S.E., Zuber J., Perrimon N. (2017) Loss-of-function genetic tools for animal models: cross-species and cross-platform differences. Nat. Rev. Genet. 18, 24–40.
  11. Chong Z.X., Yeap S.K., Ho W.Y. (2021) Transfection types, methods and strategies: a technical review. Peer J. 9, e11165.
  12. Harper J.W., Schulman B.A. (2021) Cullin-RING ubiquitin ligase regulatory circuits: a quarter century beyond the F-box hypothesis. Annu. Rev. Biochem. 90, 403‒429.
  13. Sun J., Deng W.-M. (2005) Notch-dependent downregulation of the homeodomain gene cut is required for the mitotic cycle/endocycle switch and cell differentiation in Drosophila follicle cells. Development. 132, 4299–4308.
  14. Knapp E.M., Li W., Sun J. (2019) Downregulation of homeodomain protein Cut is essential for Drosophila follicle maturation and ovulation. Development. 146, dev179002.
  15. Bassett A.R., Tibbit C., Ponting C.P., Liu J.-L. (2014) Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9. Biol. Open. 3, 42–49.
  16. Niki Y., Yamaguchi T., Mahowald A.P. (2006) Establishment of stable cell lines of Drosophila germ-line stem cells. Proc. Natl. Acad. Sci. USA. 103, 16325–16330.
  17. Gratz S.J., Ukken F.P., Rubinstein C.D., Thiede G., Donohue L.K., Cummings A.M., OꞌConnor-Giles K.M. (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics. 196, 961–971.
  18. Kunzelmann S., Böttcher R., Schmidts I., Förstemann K. (2016) A comprehensive toolbox for genome editing in cultured Drosophila melanogaster cells. G3: Genes, Genomes, Genetics. 6, 1777–1785.
  19. Böttcher R., Hollmann M., Merk K., Nitschko V., Obermaier C., Philippou-Massier J., Wieland I., Gaul U., Förstemann K. (2014) Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. Nucl. Acids Res. 42, e89–e89.
  20. Perkins L.A., Holderbaum L., Tao R., Hu Y., Sopko R., McCall K., Yang-Zhou D., Flockhart I., Binari R., Shim H.S., Miller A., Housden A., Foos M., Randkelv S., Kelley C., Namgyal P., Villalta C., Liu L.P., Jiang X., Huan-Huan Q., Wang X., Fujiyama A., Toyoda A., Ayers K., Blum A., Czech B., Neumuller R., Yan D., Cavallaro A., Hibbard K., Hall D., Cooley L., Hannon G.J., Lehmann R., Parks A., Mohr S.E., Ueda R., Kondo S., Ni J.Q., Perrimon N. (2015) The transgenic RNAi project at Harvard medical school: resources and validation. Genetics. 201, 843–852.
  21. Sytnikova Y.A., Rahman R., Chirn G.-W., Clark J.P., Lau N.C. (2014) Transposable element dynamics and PIWI regulation impacts lncRNA and gene expression diversity in Drosophila ovarian cell cultures. Genome Res. 24, 1977–1990.
  22. Stoyko D., O T., Hernandez A., Konstantinidou P., Meng Q., Haase A.D. (2022) CRISPR-Cas9 genome editing and rapid selection of cell pools. Curr. Protoc. 2, e624.
  23. Xia B., Amador G., Viswanatha R., Zirin J., Mohr S.E., Perrimon N. (2020) CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells. Nat. Protoc. 15, 3478–3498.

Arquivos suplementares

Arquivos suplementares
Ação
1. JATS XML
Baixar
2. Fig. 1. Results of Cul3 and EloB gene knockout in OSC. Changes in the expression level of Cul3 and EloB genes in knockout cells relative to the original OSC[Cas9+] line (a). The expression level measured by qRT-PCR was normalized to rp49 expression. The average measurement values ​​for two biological replicates with three technical replicates each are shown; the measurement errors are shown as the standard error of the mean. The statistical significance of differences was assessed using the Wilcoxon method. PCR analysis of the genome of Cul3-KO (b) and EloB-KO (c) cells revealed the presence of both wild-type alleles and a mutant allele with an insertion of the hyg gene in both lines. The arrangement of the primers used for the gene sequence, the size of the amplicons, and the PCR result (electrophoresis in an agarose gel) are shown.

Baixar (150KB)
Metadados
3. Fig. 2. Scheme of construction of donor plasmid pAc-MT-shRNA-bsd containing MT-shRNA-bsd cassette with inducible shRNA (a). Detailed description is presented in the text and Experimental section. puro_R and puro_L are fragments of puro gene in pAc-MT-shRNA-bsd which will serve as arms for homologous recombination. MT-shRNA-bsd cassette from donor plasmid is introduced into pAc-sgRNA-Cas9 transgene preliminarily integrated into genome (its part with puro and Cas9 genes is shown) using Cas9 and is activated by addition of copper ions (b). Expressed shRNAs are processed to siRNAs which in complex with Drosophila Ago2 protein bind to complementary mRNA and cause its cleavage by RNAi mechanism.

Baixar (288KB)
Metadados
4. Fig. 3. Changes in the expression level of the Cul3 (a) and cut (b) genes upon addition of copper ions at concentrations of 0.5, 1, and 2 mM to cell lines in which cassettes with the corresponding shRNAs were introduced into the genome, compared to expression in OSC[Cas9+]. The expression level measured by qRT-PCR was normalized to the expression of rp49. Shown are the average values ​​of measurements for two to three biological replicates with three technical replicates in each; measurement errors are shown as standard errors of the mean. The statistical significance of differences was assessed using the Wilcoxon method; ns ‒ differences in means are statistically insignificant.

Baixar (137KB)
Metadados

Declaração de direitos autorais © Russian Academy of Sciences, 2024