Phantom Database for co-silencing genes
In the following are artificial microRNA (amiRNA) files that were developed in Hauser, F et al. A genomic-scale artificial microRNA library as a tool to investigate the functionally redundant gene space in Arabidopsis. Plant Cell vol 25,8 (2013): 2848-64. and Hauser, F, Ceciliato PHO et al. A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO2 and ABA responses. Journal of Experimental Botany vol. 70,2 (2019): 641-651.
14,000 transformed T3 generation seeds expressing amiRNAs from these libraries are described in Hauser, Ceciliato et al (2019). Each of the seed pools listed below targets defined classes of protein families. For the meanings of the abbreviations, please see the descriptions at the ABRC links below or see Hauser, Ceciliato et al (2019). The following seed libraries can be ordered in pools at ABRC at these links:
- CS99427: PKR library,
- CS99428: BNO library,
- CS99429: CSI library,
- CS99430: TFB library,
- CS99431: TEC library,
- CS99432: PEC library,
- CS99433: UNC library,
- CS99434: DMF library,
- CS99435: HEC library,
- CS99436: TRP library.
- CS886808: additional TRP library.
The following excel file lists these 22,000 amiRNAs and the combinations of genes targeted by each amiRNA. There are 59901 rows in this table because the table is listed by genes (AGI numbers) targeted by the 22,000 amiRNAs, rather than by the 22,000 amiRNAs.
Furthermore, the WMD3 tool can also be used to identify potential target genes from an isolated amiRNA sequence.
Ten amiRNA libraries were synthesized containing 22,000 total distinct plasmids containing amiRNAs as described in Hauser et al (2013). These amiRNA libraries can be used to screen for functionally redundant genes as described in Hauser, Ceciliato et al (2019). 96% of these amiRNAs are designed to target 2 to 5 genes, meaning that identification of the responsible genes for a phenotype should be facile. These cDNA libraries can be ordered from ABRC at these links:
- CD4-62: PKR library (20mer amiRNAs),
- CD4-63: BNO library (20mer amiRNAs),
- CD4-64: CSI library (20mer amiRNAs),
- CD4-65: TFB library (20mer amiRNAs),
- CD4-66: TEC library (20mer amiRNAs),
- CD4-67: PEC library (20mer amiRNAs),
- CD4-68: UNC library (20mer amiRNAs),
- CD4-69: DMF library (20mer amiRNAs),
- CD4-70: HEC library (20mer amiRNAs),
- CD4-71: TRP library (20mer amiRNAs),
- CD4-72: PKR library (21mer amiRNAs),
- CD4-73: BNO library (21mer amiRNAs),
- CD4-74: CSI library (21mer amiRNAs),
- CD4-75: TFB library (21mer amiRNAs),
- CD4-76: TEC library (21mer amiRNAs),
- CD4-77: PEC library (21mer amiRNAs),
- CD4-78: UNC library (21mer amiRNAs),
- CD4-79: DMF library (21mer amiRNAs),
- CD4-80: HEC library (21mer amiRNAs),
- CD4-81: TRP library (21mer amiRNAs),
- CD4-82: Set of 20 PHANTOM amiRNA pools (CD4-62 to CD4-81).
Hauser et al. (2013) computed over 2,000,000 amiRNAs that are predicted to co-silence closely related genes. The following file link lists the >2,000,000 amiRNAs and their predicted target genes in Arabidopsis thaliana. This link is seachable either with an amiRNA sequence or with gene identifier numbers, to identify amiRNAs that can target gene combinations of interest for reverse genetics.
Small Proof of Concept amiRNA Library for Reverse Genetics
ReferencesTitle: A Genomic-Scale Artificial MicroRNA Library as a Tool to Investigate the Functionally Redundant Gene Space in Arabidopsis
Authors: Felix Hauser, Wenxiao Chen, Ulrich Deinlein, Kenneth Chang, Stephan Ossowski, Joffrey Fitz, Gregory J. Hannon, Julian I. Schroeder
Abstract: Traditional forward genetic screens are limited in the identification of homologous genes with overlapping functions. Here, we report the analyses and assembly of genome-wide protein family definitions that comprise the largest estimate for the potentially redundant gene space in Arabidopsis thaliana. On this basis, a computational design of genome-wide family-specific artificial microRNAs (amiRNAs) was performed using high-performance computing resources. The amiRNA designs are searchable online (http://phantomdb.ucsd.edu). A computationally derived library of 22,000 amiRNAs was synthesized in 10 sublibraries of 1505 to 4082 amiRNAs, each targeting defined functional protein classes. For example, 2964 amiRNAs target annotated DNA and RNA binding protein families and 1777 target transporter proteins, and another sublibrary targets proteins of unknown function. To evaluate the potential of an amiRNA-based screen, we tested 122 amiRNAs targeting transcription factor, protein kinase, and protein phosphatase families. Several amiRNA lines showed morphological phenotypes, either comparable to known phenotypes of single and double/triple mutants or caused by overexpression of microRNAs. Moreover, novel morphological and abscisic acid–insensitive seed germination mutants were identified for amiRNAs targeting zinc finger homeodomain transcription factors and mitogen-activated protein kinase kinase kinases, respectively. These resources provide an approach for genome-wide genetic screens of the functionally redundant gene space in Arabidopsis.
Title: A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO2 and ABA responses
Authors: Hauser, F., Ceciliato, P., Lin, Y., Guo, D., Gregerson, J., Abbasi, N., Youhanna, D., Park, J., Dubeaux, G., Shani, E., Poomchongkho, N., Schroeder, J.
Abstract: The identification of homologous genes with functional overlap in forward genetic screens is severely limited. Here, we report the generation of over 14000 artificial microRNA (amiRNA)-expressing plants that enable screens of the functionally redundant gene space in Arabidopsis. A protocol was developed for isolating robust and reproducible amiRNA mutants. Examples of validation approaches and essential controls are presented for two new amiRNA mutants that exhibit genetically redundant phenotypes and circumvent double mutant lethality. In a forward genetic screen for abscisic acid (ABA)-mediated inhibition of seed germination, amiRNAs that target combinations of known redundant ABA receptor and SnRK2 kinase genes were rapidly isolated, providing a strong proof of principle for this approach. A new ABA-insensitive amiRNA line that targets three avirulence-induced gene 2(-like) genes was isolated . A thermal imaging screen for plants with impaired stomatal opening in response to low CO2 exposure led to the isolation of a new amiRNA targeting two essential proteasomal subunits, PAB1 and PAB2. The seed library of 11000 T2 amiRNA lines (with 3000 lines in progress) generated here provides a new platform for forward genetic screens and has been made available to the Arabidopsis Biological Resource Center (ABRC). Optimized procedures for amiRNA screening and controls are described.