Target Gene Identification and sgRNA Design for Waterlogging Tolerance in Foxtail Millet via CRISPR-based Transcriptional Activation

Background: CRISPR activation (CRISPRa) uses non-functional Cas9 endonuclease (dCas9) but retains the genome targeting ability through its single guide RNAs (sgRNAs). CRISPRa is widely utilized as a gene activation system exploiting its ability to recruit various transcriptional activation domains (TADs) to enhance the expression of the target gene(s). Drought tolerant and resource-efficient crops like millets can mitigate the effects of climate change and strengthen food security. Objective: This study aimed to use the Setaria italica (foxtail millet) genome sequence to identify a target gene and the subsequent generation of sgRNAs for use in CRISPRa for conferring waterlogging tolerance that will benefit the future expansion of its cultivation area. Methods and Results: Leveraging existing RNA-seq data and information on functional studies in model plants and from other cereal species, maize and barley, have enabled the identification of candidate ERFVII from the foxtail millet genome sequence in the attempt to engineer waterlogging tolerance. The study provides a step-by-step example for using publicly accessible databases and bioinformatics tools from NCBI and Phytozome to identify and characterize the ortholog from Setaria italica. Softberry was used for promoter annotation to obtain the transcription start site (TSS). Subsequently, CRISP-P 2.0 design tools were employed to generate and select a few efficient sgRNAs for CRISPRa that minimize potentially deleterious off-target binding. Conclusion: The study is a helpful example of how to advance in genomics research, including the revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species by utilizing the available genome sequence.

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De Novo Genome Assembly of a Foxtail Millet Cultivar Huagu11 Uncovered the Genetic Difference to the Cultivar Yugu1, and the Genetic Mechanism of Imazethapyr Tolerance

10.21203/rs.3.rs-143813/v1 ◽

2021 ◽

Author(s):

Jie Wang ◽

Shiming Li ◽

Lei Lan ◽

Mushan Xie ◽

Shu Cheng ◽

...

Keyword(s):

Next Generation Sequencing ◽

Genome Sequence ◽

Reference Genome ◽

Foxtail Millet ◽

Setaria Italica ◽

Genome Comparison ◽

Sequencing Technology ◽

High Quality ◽

Next Generation Sequencing Technology ◽

Generation Sequencing

Abstract Background: Setaria italica is the second-most widely planted species of millets in the world and an important model grain crop for the research of C4 photosynthesis and abiotic stress tolerance. Through three genomes assembly and annotation efforts, all genomes were based on next generation sequencing technology, which limited the genome continuity. Results: Here we report a high-quality whole-genome of new cultivar Huagu11, using single-molecule real-time sequencing and High-throughput chromosome conformation capture (Hi-C) mapping technologies. The total assembly size of the Huagu11 genome was 408.37 Mb with a scaffold N50 size of 45.89 Mb. Compared with the other three reported millet genomes based on the next generation sequencing technology, the Huagu11 genome had the highest genomic continuity. Intraspecies comparison showed about 94.97% and 94.66% of the Yugu1 and Huagu11 genomes, respectively, were able to be aligned as one-to-one blocks with four chromosome inversion. The Huagu11 genome contained approximately 19.43 Mb Presence/absence Variation (PAV) with 627 protein-coding transcripts, while Yugu1 genomes had 20.53 Mb PAV sequences encoding 737 proteins. Overall, 969,596 Single-nucleotide polymorphism (SNPs) and 156,282 insertion-deletion (InDels) were identified between these two genomes. The genome comparison between Huagu11 and Yugu1 should reflect the genetic identity and variation between the cultivars of foxtail millet to a certain extent. The Ser-626-Aln substitution in acetohydroxy acid synthase (AHAS) was found to be relative to the imazethapyr tolerance in Huagu11. Conclusions: A new improved high-quality reference genome sequence of Setaria italica was assembled, and intraspecies genome comparison determined the genetic identity and variation between the cultivars of foxtail millet. Based on the genome sequence, it was found that the Ser-626-Aln substitution in AHAS was responsible for the imazethapyr tolerance in Huagu11. The new improved reference genome of Setaria italica will promote the genic and genomic studies of this species and be beneficial for cultivar improvement.

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A Remorin Gene SiREM6, the Target Gene of SiARDP, from Foxtail Millet (Setaria italica) Promotes High Salt Tolerance in Transgenic Arabidopsis

PLoS ONE ◽

10.1371/journal.pone.0100772 ◽

2014 ◽

Vol 9 (6) ◽

pp. e100772 ◽

Cited By ~ 16

Author(s):

Jing Yue ◽

Cong Li ◽

Yuwei Liu ◽

Jingjuan Yu

Keyword(s):

Salt Tolerance ◽

Target Gene ◽

Transgenic Arabidopsis ◽

Foxtail Millet ◽

High Salt ◽

Setaria Italica ◽

High Salt Tolerance

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Mutational analysis of the DNA binding, dimerization, and transcriptional activation domains of MEF2C.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2627 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2627-2636 ◽

Cited By ~ 130

Author(s):

J D Molkentin ◽

B L Black ◽

J F Martin ◽

E N Olson

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Mutational Analysis ◽

Gene Activation ◽

Positive Control ◽

Dominant Negative ◽

Activation Domains ◽

Muscle Gene Expression ◽

Transcriptional Activation Domains ◽

Muscle Gene

There are four members of the myocyte enhancer factor 2 (MEF2) family of transcription factors in vertebrates, MEF2A, -B, -C, and -D, which have homology within a MADS box at their amino termini and an adjacent motif known as the MEF2 domain. These factors activate muscle gene expression by binding as homo- and heterodimers to an A/T-rich DNA sequence in the control regions of muscle-specific genes. To understand the mechanisms of muscle gene activation of MEF2 factors, we generated a series of deletion and site-directed mutants of MEF2C. These mutants demonstrated that the MADS and MEF2 domains mediate DNA binding and dimerization, whereas the carboxyl terminus is required for transcriptional activation. Amino acids that are essential for MEF2 site-dependent transcription but which do not affect DNA binding were also identified in the MEF2 domain. This type of positive-control mutant demonstrates that the transcription activation domain of MEF2C, although separate from the MEF2 domain, is dependent on this domain for transcriptional activation through the MEF2 site. MEF2 mutants that are defective for DNA binding act as dominant negative mutants and can inhibit activation of MEF2-dependent genes by wild-type MEF2C.

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Genome sequence of foxtail millet (Setaria italica) provides insights into grass evolution and biofuel potential

Nature Biotechnology ◽

10.1038/nbt.2195 ◽

2012 ◽

Vol 30 (6) ◽

pp. 549-554 ◽

Cited By ~ 345

Author(s):

Gengyun Zhang ◽

Xin Liu ◽

Zhiwu Quan ◽

Shifeng Cheng ◽

Xun Xu ◽

...

Keyword(s):

Genome Sequence ◽

Foxtail Millet ◽

Setaria Italica ◽

Grass Evolution

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Multiplexed and tunable transcriptional activation by promoter insertion using nuclease-assisted vector integration

Nucleic Acids Research ◽

10.1093/nar/gkz210 ◽

2019 ◽

Vol 47 (12) ◽

pp. e67-e67 ◽

Cited By ~ 3

Author(s):

Alexander Brown ◽

Jackson Winter ◽

Michael Gapinske ◽

Nathan Tague ◽

Wendy S Woods ◽

...

Keyword(s):

Transcriptional Activation ◽

Target Gene ◽

Gene Activation ◽

Precise Control ◽

Vector Integration ◽

Synthetic Promoters ◽

Artificial Transcription Factors ◽

A Genome ◽

Control Of Expression ◽

Line Engineering

Abstract The ability to selectively regulate expression of any target gene within a genome provides a means to address a variety of diseases and disorders. While artificial transcription factors are emerging as powerful tools for gene activation within a natural chromosomal context, current generations often exhibit relatively weak, variable, or unpredictable activity across targets. To address these limitations, we developed a novel system for gene activation, which bypasses native promoters to achieve unprecedented levels of transcriptional upregulation by integrating synthetic promoters at target sites. This gene activation system is multiplexable and easily tuned for precise control of expression levels. Importantly, since promoter vector integration requires just one variable sgRNA to target each gene of interest, this procedure can be implemented with minimal cloning. Collectively, these results demonstrate a novel system for gene activation with wide adaptability for studies of transcriptional regulation and cell line engineering.

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De novo genome assembly of a foxtail millet cultivar Huagu11 uncovered the genetic difference to the cultivar Yugu1, and the genetic mechanism of imazethapyr tolerance

BMC Plant Biology ◽

10.1186/s12870-021-03003-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jie Wang ◽

Shiming Li ◽

Lei Lan ◽

Mushan Xie ◽

Shu Cheng ◽

...

Keyword(s):

Next Generation Sequencing ◽

Genome Sequence ◽

Reference Genome ◽

Foxtail Millet ◽

Setaria Italica ◽

Genome Comparison ◽

Sequencing Technology ◽

High Quality ◽

Next Generation Sequencing Technology ◽

Generation Sequencing

Abstract Background Setaria italica is the second-most widely planted species of millets in the world and an important model grain crop for the research of C4 photosynthesis and abiotic stress tolerance. Through three genomes assembly and annotation efforts, all genomes were based on next generation sequencing technology, which limited the genome continuity. Results Here we report a high-quality whole-genome of new cultivar Huagu11, using single-molecule real-time sequencing and High-throughput chromosome conformation capture (Hi-C) mapping technologies. The total assembly size of the Huagu11 genome was 408.37 Mb with a scaffold N50 size of 45.89 Mb. Compared with the other three reported millet genomes based on the next generation sequencing technology, the Huagu11 genome had the highest genomic continuity. Intraspecies comparison showed about 94.97 and 94.66% of the Yugu1 and Huagu11 genomes, respectively, were able to be aligned as one-to-one blocks with four chromosome inversion. The Huagu11 genome contained approximately 19.43 Mb Presence/absence Variation (PAV) with 627 protein-coding transcripts, while Yugu1 genomes had 20.53 Mb PAV sequences encoding 737 proteins. Overall, 969,596 Single-nucleotide polymorphism (SNPs) and 156,282 insertion-deletion (InDels) were identified between these two genomes. The genome comparison between Huagu11 and Yugu1 should reflect the genetic identity and variation between the cultivars of foxtail millet to a certain extent. The Ser-626-Aln substitution in acetohydroxy acid synthase (AHAS) was found to be relative to the imazethapyr tolerance in Huagu11. Conclusions A new improved high-quality reference genome sequence of Setaria italica was assembled, and intraspecies genome comparison determined the genetic identity and variation between the cultivars of foxtail millet. Based on the genome sequence, it was inferred that the Ser-626-Aln substitution in AHAS was responsible for the imazethapyr tolerance in Huagu11. The new improved reference genome of Setaria italica will promote the genic and genomic studies of this species and be beneficial for cultivar improvement.

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Pygo2 Associates with MLL2 Histone Methyltransferase and GCN5 Histone Acetyltransferase Complexes To Augment Wnt Target Gene Expression and Breast Cancer Stem-Like Cell Expansion

Molecular and Cellular Biology ◽

10.1128/mcb.00465-10 ◽

2010 ◽

Vol 30 (24) ◽

pp. 5621-5635 ◽

Cited By ~ 50

Author(s):

Jiakun Chen ◽

Qicong Luo ◽

Yuanyang Yuan ◽

Xiaoli Huang ◽

Wangyu Cai ◽

...

Keyword(s):

Breast Cancer ◽

Transcriptional Activation ◽

Cell Expansion ◽

Breast Cancer Cells ◽

Target Gene ◽

Target Genes ◽

Histone Acetyltransferase ◽

Gene Activation ◽

Histone Methyltransferase ◽

T Cell Factor

ABSTRACT Resent studies have identified Pygopus as a core component of the β-catenin/T-cell factor (TCF)/lymphoid-enhancing factor 1 (LEF) transcriptional activation complex required for the expression of canonical Wg/Wnt target genes in Drosophila. However, the biochemical involvement of mammalian Pygopus proteins in β-catenin/TCF/LEF gene activation remains controversial. In this study, we perform a series of molecular/biochemical experiments to demonstrate that Pygo2 associates with histone-modifying enzymatic complexes, specifically the MLL2 histone methyltransferase (HMT) and STAGA histone acetyltransferase (HAT) complexes, to facilitate their interaction with β-catenin and to augment Wnt1-induced, TCF/LEF-dependent transcriptional activation in breast cancer cells. We identify a critical domain in Pygo2 encompassing the first 47 amino acids that mediates its HMT/HAT interaction. We further demonstrate the importance of this domain in Pygo2's ability to transcriptionally activate both artificial and endogenous Wnt target genes and to expand breast cancer stem-like cells in culture. This work now links mechanistically Pygo2's role in histone modification to its enhancement of the Wnt-dependent transcriptional program and cancer stem-like cell expansion.

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Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells

10.1101/491894 ◽

2018 ◽

Author(s):

Onur Tidin ◽

Elias T. Friman ◽

Felix Naef ◽

David M. Suter

Keyword(s):

Transcriptional Activation ◽

Target Gene ◽

Target Genes ◽

Nuclear Translocation ◽

Gene Activation ◽

Transcriptional Response ◽

Tissue Growth ◽

Live Cells ◽

High Temporal Resolution ◽

Transcriptional Responses

AbstractThe transduction of extracellular signals through signaling pathways that culminate in a transcriptional response is central to many biological processes. However, quantitative relationships between activities of signaling pathway components and transcriptional output of target genes remain poorly explored. Here we developed a dual bioluminescence imaging strategy allowing simultaneous monitoring of nuclear translocation of the SMAD4 and SMAD2 transcriptional activators upon TGF-β stimulation, and the transcriptional response of the endogenous connective tissue growth factor (ctgf) gene. Using cell lines allowing to vary exogenous SMAD4/2 expression levels, we performed quantitative measurements of the temporal profiles of SMAD4/2 translocation and ctgf transcription kinetics in hundreds of individual cells at high temporal resolution. We found that while nuclear translocation efficiency had little impact on initial ctgf transcriptional activation, high total cellular SMAD4 but not SMAD2 levels increased the probability of cells to exhibit a sustained ctgf transcriptional response. The approach we present here allows time-resolved single cell quantification of transcription factor dynamics and transcriptional responses and thereby sheds light on the quantitative relationship between SMADs and target gene responses.

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