Abstract
Novel tools and methods for regulating in vivo plant gene expression are quickly gaining popularity and utility due to recent advances in CRISPR-dCas9 chimeric effector regulators, otherwise known as CRISPR artificial transcription factors (CRISPR-ATFs). These tools are especially useful for studying gene function and interaction within various regulatory networks. First generation CRISPR-ATFs are nuclease-deactivated (dCas9) CRISPR systems where dCas9 proteins are fused to known transcriptional activator domains (VP64) or repressor domains (SRDX). When multiple chimeric dCas9-effector fusions are guided to gene regulatory regions via CRISPR gRNAs, they can modulate expression of transcript levels in planta. The protocol presented here provides a detailed procedure for activating AtPAP1 and repressing AtCSTF64 in Arabidopsis thaliana. This protocol makes use of our plant CRISPR toolbox to streamline the assembly and cloning of multiplex CRISPR-Cas9 transcriptional regulatory constructs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Choo Y, Isalan M (2000) Advances in zinc finger engineering. Curr Opin Struct Biol 10:411–416
Sanchez J-P, Ullman C, Moore M, Choo Y, Chua N-H (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol 43(12):1465–1472
Sera T (2009) Zinc-finger-based artificial transcription factors and their applications. Adv Drug Deliv Rev 61:513–526
Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326(5959):1509–1512. doi:10.1126/science.1178811
Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326(5959):1501. doi:10.1126/science.1178817
Mahfouz MM, Li L, Piatek M, Fang X, Mansour H, Bangarusamy DK, Zhu JK (2012) Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein. Plant Mol Biol 78(3):311–321. doi:10.1007/s11103-011-9866-x
Perez-Pinera P, Ousterout D, Brunger J, Farin A, Glass K, Guilak F, Crawford G, Hartemink A, Gersback C (2013) Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat Methods 10(3):239–242
Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451. doi:10.1016/j.cell.2013.06.044
Maeder M, Linder S, Cascio V, Fu Y, Ho Q, Joung K (2013) CRISPR RNA-guided activation of endogenous human genes. Nat Methods. doi:10.1038/NMETH.2598
Chavez A, Tuttle M, Pruitt B, Ewen-Campen B, Chari R, Ter-Ovanesyan D, Haque S, Cecchi R, Kowal E, Buchthal J, Housden B, Perrimon N, Collins J, Church G (2016) Comparison of Cas9 activators in multiple species. Nat Methods. doi:10.1038/NMETH.3871
Aoyama T, Chua N-H (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11(3):605–612
Uesugi M, Nyanguile O, Lu H, Levine A, Verdine G (1997) Induced α helix in the VP16 activation domain upon binding to a human TAF. Science 277:1310–1313
Carey M, Lin Y-S, Green M, Ptashne M (1990) A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives. Nature 345:361–364
Perez-Pinera P, Kocak D, Vockley C, Adler A, Kabadi A, Polstein L, Thakore P, Glass K, Ousterout D, Leong K, Guilak F, Crawford G, Reddy T, Gersback C (2013) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods. doi:10.1038/NMETH.2600
Hiratsu K, Matsui K, Koyama T, Ohme-Takagi M (2003) Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J 34:733–739
Piatek A, Ali Z, Baazim H, Li L, Abulfaraj A, Al-Shareef S, Aouida M, Mahfouz M (2015) RNA-guided transcriptional regulation in Planta via synthetic dCas9-based transcription factors. Plant Biotechnol J 13:578–589
Szemenyei H, Hannon M, Long J (2008) TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science 319:1384–1386
Lowder L, Zhang D, Baltes N, Paul J III, Tang X, Zheng X, Voytas D, Hsieh T-F, Zhang Y, Qi Y (2015) A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 169:971–985
Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108
Isaac S, Jiang F, Doudna J, Lim W, Narlikar G, Almeida R (2016) Nucleosome breathing and remodeling constrain CRISPR-Cas9 function. elife 6(5). doi:10.7554/eLife.13450
Horlbeck MA, Witkowsky LB, Guglielmi B, Replogle JM, Gilbert LA, Villalta JE, Torigoe SE, Tjian R, Weissman JS (2016) Nucleosomes impede Cas9 access to DNA in vivo and in vitro. elife 5. doi:10.7554/eLife.12677
Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12(12):2383–2394
Liu F, Marquardt S, Lister C, Swiezewski S, Dean C (2010) Targeted 3′ processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing. Science 327(5961):94–97. doi:10.1126/science.1180278
Graham D, Root D (2015) Resources for the design of CRISPR gene editing experiments. Genome Biol 16:260
Acknowledgments
This work is supported by a Collaborative Funding Grant (2016-CFG-8003) from North Carolina Biotechnology Center and Syngenta Biotechnology to Y.Q.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Lowder, L.G., Paul, J.W., Qi, Y. (2017). Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems. In: Kaufmann, K., Mueller-Roeber, B. (eds) Plant Gene Regulatory Networks. Methods in Molecular Biology, vol 1629. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7125-1_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7125-1_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7124-4
Online ISBN: 978-1-4939-7125-1
eBook Packages: Springer Protocols