Transcriptional Activation Domain Research Articles

A novel variant of GLI3, p.Asp1514Thrfs*5, is identified in a Chinese family affected by polydactyly.

BackgroundPolydactyly is a common congenital malformation characterized by the presence of supernumerary fingers or toes. In this case study, we sought to identify the causative pathogenic factor in a family from a northern region of China affected by non‐syndromic postaxial polydactyly (PAP).MethodsAfter recruiting a three‐generation family with PAP, whole‐exome sequencing was performed to identify the causative variant. In silico analysis and Sanger sequencing were used to validate the variant.ResultsWe identified a novel heterozygous frameshift variant (NM_000168.6:c.4540delG, p.Asp1514Thrfs*5) in the transcriptional activator (TA1) domain of the GLI3 gene.ConclusionThe novel frameshift variant identified in this study further confirms the relationship between non‐syndromic PAP and GLI3 and extends the previously established mutational and phenotypic spectra of GLI3.

Directed mutational scanning reveals a balance between acidic and hydrophobic residues in strong human activation domains.

SUMMARYAcidic activation domains are intrinsically disordered regions of the transcription factors that bind coactivators. The intrinsic disorder and low evolutionary conservation of activation domains have made it difficult to identify the sequence features that control activity. To address this problem, we designed thousands of variants in seven acidic activation domains and measured their activities with a high-throughput assay in human cell culture. We found that strong activation domain activity requires a balance between the number of acidic residues and aromatic and leucine residues. These findings motivated a predictor of acidic activation domains that scans the human proteome for clusters of aromatic and leucine residues embedded in regions of high acidity. This predictor identifies known activation domains and accurately predicts previously unidentified ones. Our results support a flexible acidic exposure model of activation domains in which the acidic residues solubilize hydrophobic motifs so that they can interact with coactivators. A record of this paper’s transparent peer review process is included in the supplemental information.

The role of the NF-kappa B transcription activation domain in specific and nonspecific DNA interactions

The transcription factor NFκB activates genes that mediate inflammation, the immune response, and cell death and survival via interactions with both DNA and co-activator proteins. NFκB dimers (p50/RelA) contain a natively folded Rel-homology domain (RHD) and an intrinsically disordered transcription activation domain (TAD). Historically, the RHD was believed to mediate DNA binding whereas the TAD was responsible for the protein-protein interactions necessary for transcription activation. Nearly all previous in vitro studies have used truncated NFκB constructs and studied the roles of these domains in isolation.

CRISPR-Act3.0-Based Highly Efficient Multiplexed Gene Activation in Plants.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)-mediated genome editing has revolutionized fundamental research and plant breeding. Beyond gene editing, CRISPR/Cas systems have been repurposed as a platform for programmable transcriptional regulation. Catalytically inactive Cas variants (dCas), when fused with transcriptional activation domains, allow for specific activation of any target gene in the genome without inducing DNA double-strand breaks. CRISPR activation enables simultaneous activation of multiple genes, holding great promise in the identification of gene regulatory networks and rewiring of metabolic pathways. Here, we describe a simple protocol for constructing a dCas9-mediated multiplexed gene activation system based on the CRISPR-Act3.0 system. The resulting vectors are tested in rice protoplasts. © 2022 Wiley Periodicals LLC. Basic Protocol 1: sgRNA design and construction of CRISPR-Act3.0 vectors for multiplexed gene activation Basic Protocol 2: Determining the activation efficiency of CRISPR-Act3.0 vectors using rice protoplasts.

A Potential MDM2 Inhibitor Formed by Restoring the Native Conformation of the p53 α-Helical Peptide on Gold Nanoparticles.

Many efforts have been made to develop inhibitors of MDM2 as potential drugs for cancer therapy. In this work, we use our previous developed conformational engineering technique to stabilize the binding conformation of the p53 transcription activation domain (TAD) peptide on gold nanoparticles (AuNPs), and create an AuNP-based anti-MDM2 artificial antibody, denoted as anti-MDM2 Goldbody, that specifically binds MDM2. Though the free TAD peptide is unstructured, circular dichroism (CD) spectra confirm that its α-helical conformation in the original p53 protein is restored on the anti-MDM2 Goldbody, and surface plasmon resonance (SPR) experiments confirm that there is strong specific interaction between the anti-MDM2 Goldbody and MDM2, demonstrating the anti-MDM2 Goldbody as a potential inhibitor of MDM2. This work demonstrates that the conformational engineering technique is not limited to the antigen-antibody systems, but can also be applied more widely in other protein-protein interfaces to create increasingly more artificial proteins for various biomedical applications.

The kinetic landscape of human transcription factors

Progress in genomics has enabled mapping where in the genome Transcription Factors (TFs) bind, yet what TFs do once bound remains poorly understood. One reason is that the transcription activation domains of TFs are constituted of poorly conserved intrinsically disordered domains, limiting the use of classic biochemistry approaches or the identification of conserved motifs. Single-molecule studies of transcription dynamics in living cells are capable of providing unique functional insights into TF activity, but are often limited in throughput.

Uncovering the Distinct Properties of a Bacterial Type I-E CRISPR Activation System.

Synthetic gene regulators based upon CRISPR-Cas systems offer programmable technologies to control gene expression in bacteria. Bacterial CRISPR activators (CRISPRa) have been developed that use engineered type II CRISPR-dCas9 to localize transcription activation domains near promoter elements. However, several reports have demonstrated distance-dependent requirements and periodical activation patterns that overall limit their flexibility. Here, we demonstrate the potential of using an alternative type I-E CRISPR-Cas system to create a CRISPRa with distinct and expanded regulatory properties. Furthermore, we create the first bacterial CRISPRa system based upon a type I-E CRISPR-Cas and characterize the distance-dependent activation patterns to reveal a distinct and more frequent periodicity of activation.

CRISPR-Based Transcriptional Activation in Drosophila.

Overexpression is one of the classical approaches to study pleiotropic functions of genes of interest. To achieve overexpression, we often increase the transcription by introducing genes on exogenous vectors or by using the CRISPR/dCas9-based transcriptional activation system. To date, the most efficient CRISPR/dCas9-based transcriptional activator is the Synergistic Activation Mediator (SAM) system whereby three different transcriptional activation domains are directly fused to dCas9 and MS2 phage Coat Protein (MCP), respectively, and the system in Drosophila is named flySAM. Here we describe the effective and convenient transcriptional activation system, flySAM, starting from vector construction, microinjection, and transgenic fly selection to the phenotypic analysis.

Design, Characterization, and Application of Targeted Gene Activation in Bacteria Using a Modular CRISPRa System.

CRISPR-Cas regulators have provided an excellent toolbox to control gene expression due to the versatility of its components and the easy programming of the single guide RNA (sgRNA) to target DNA sequences. Included in this are CRISPR activation (CRISPRa) systems. These systems allow users to activate transcription of a target gene through the localization of transcription activation domains (ADs) near promoter elements, which in turn recruit RNA polymerase (RNAP) to turn on transcription. A variety of different CRISPRa systems have been described that vary in AD type, recruitment strategies, and CRISPR-Cas systems. Recently, a highly modular CRISPRa system was described that allows for facile exchange of ADs and CRISPR-Cas components. This allows for the creation of CRISPRa systems with unique properties, for example, ability to activate from specific positions upstream of a gene of interest. Here, we describe a protocol for designing, characterizing, and applying the modular CRISPRa system for gene activation in E. coli. We first focus on how to identify activating sites upstream of promoters and the cloning of the targeting sgRNA. We then describe how to perform a fluorescence experiment to evaluate activation of a single target site. Finally, we explain how to adapt the system to expand the target range and how to characterize the activation pattern obtained from different CRISPRa designs.

Studies on the interactions of AFPs and bZIP transcription factor ABI5

AFP1 interacts with ABI5 and negatively regulates the abscisic acid (ABA) signaling by accelerating ABI5's degradation during the seed germination phase in Arabidopsis, but the underlying mechanism remains unclear. Moreover, the molecular basis of the interaction between AFP1 homologs and ABI5 has yet to be elucidated. In this study, the patterns of their interactions with ABI5 were investigated in detail. We found that AFP2/3/4 can bind two regions of ABI5, one is ABI51aa to 135aa and another is ABI5202aa to 213aa. However, AFP1 only interacts with the second region of ABI5, i.e. ABI5202aa to 213aa. Prior research has shown that ABI51aa to 135aa is related to the transcriptional activity of ABI5. Thus, our results suggest that AFPs may also modulate ABI5, by directly binding to its transcriptional activation domain, thereby influencing its transcriptional activity. Further, interactions between AFPs and ABI5 were not affected if the Ser42th in the ABI5-SnRK2 motif were mutated respectively to Glu or Ala. Nevertheless, interactions between AFPs and ABI5 were eliminated if the Thr47th and Thr206th of ABI5 were mutated respectively to Glu or Ala. Since the two residues of Thr47th and Thr206th were located in the phosphorylation motifs of CKII, AFPs might regulate the activities of ABI5 transcription factor through a CKII-dependent pathway.

Duck Plague Virus pUL48 Protein Activates the Immediate-Early Gene to Initiate the Transcription of the Virus Gene

Duck plague caused by the duck plague virus (DPV) is an infectious disease that seriously harms the waterfowl breeding industry. The VP16 protein of α herpesvirus can bind to specific cis-acting elements upstream of the promoter of the immediate-early (IE, α) gene to promote the transcription of the IE gene, so it is also called the trans-inducer of IE gene (α-TIF). However, no studies on DPV α-TIF have been reported. This study investigated the DPV pUL48, a homolog of HSV-1 VP16, transcriptional activation region, target sequence, and viral protein affecting its transcriptional activation using a dual-luciferase reporter gene detection system, and pUL48 was identified as the α-TIF of DPV. (1) The regulation of pUL48 on DPV different gene promoters showed that pUL48 could activate all the promoters of IE genes (ICP4, ICP22, and ICP27) but not the promoters of early and late genes. (2) The activity of pUL48 to ICP4 and ICP22 promoters with different upstream lengths showed that pUL48 activated ICP4 and ICP22 promoters by acting on TAATGA (T) TAT element upstream of ICP4 promoter and TAATTATAT element upstream of ICP22 promoter, respectively. (3) Transcriptional activation of IE gene by truncated proteins of different lengths at the N-terminal of pUL48 was detected. The results showed that the transcriptional activation domain of pUL48 was amino acids 1–60 at the N-terminal, and amino acids 1–20 was its core region. In addition, it was found that pUL14, pUL46, and pUL47 significantly promoted the transcriptional activation of pUL48. The effects of loss of pUL47 and its nuclear localization signal on the nuclear entry and transcriptional activation function of pUL48 were further examined. The results showed that pUL47 could promote the nuclear entry of pUL48 through its nuclear localization signal at positions 40–50 and 768–777 amino acids, thus, enhancing the transcriptional activation function of pUL48 and synergistic promotion of viral gene transcription.

Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis.

The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.

Sequence Variation in the Bovine Lipin-1 Gene (LPIN1) and Its Association with Milk Fat and Protein Contents in New Zealand Holstein-Friesian × Jersey (HF × J)-cross Dairy Cows.

Simple SummaryLipins are a family of proteins involved in lipid metabolism through their phosphatidate phosphatase and transcriptional co-activator activities. Of the three family members identified, lipin-1 is highly upregulated in the bovine mammary gland during lactation, and has been implicated in regulating milk production, albeit with inconsistent results in different breeds of cattle in studies. Accordingly, its role in regulating milk synthesis is not well understood. In this study, we used polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analyses to investigate variation in the bovine lipin-1 gene in New Zealand Holstein-Friesian × Jersey-cross dairy cows and found that variation in the sixth coding exon was associated with milk fat percentage and protein percentage. This suggests that lipin-1 regulates the synthesis of milk fat and milk protein.Lipin-1 is known to play a regulatory role in tissues that function in lipid metabolism. In dairy cows, the lipin-1 gene (LPIN1) is highly expressed in the mammary gland, but its function in milk production is less understood. In this study, we used PCR-single strand conformation polymorphism analysis to investigate sequence variation in three regions of bovine LPIN1 in New Zealand Holstein-Friesian × Jersey (HF × J)-cross dairy cows, including part of the 5′ non-coding region, the region containing the LPIN1β-spliced exon, and the sixth coding exon that encodes the putative transcriptional activating domain of the protein. No variation was found in the LPIN1β-spliced exon, but two sequence variants containing one single nucleotide polymorphism (SNP) were identified in the 5′ non-coding region and four sequence variants containing four non-synonymous SNPs were identified in the sixth coding exon. Among the three common variants of the sixth coding exon, variant C was found to be associated with an increase in milk fat percentage (presence 4.96 ± 0.034% vs. absence 4.81 ± 0.050%; p = 0.006) and milk protein percentage (presence 4.09 ± 0.017% vs. absence 3.99 ± 0.025%; p = 0.001), but no associations (p > 0.01) were detected for milk yield. These results suggest that variation in LPIN1 affect the synthesis of fat and proteins in milk and has potential as a gene-marker to improve milk production traits.

Functional consequences of TCF4 missense substitutions associated with Pitt-Hopkins syndrome, mild intellectual disability, and schizophrenia

Transcription factor 4 (TCF4) is a basic helix-loop-helix transcription factor essential for neurocognitive development. The aberrations in TCF4 are associated with neurodevelopmental disorders including schizophrenia, intellectual disability, and Pitt-Hopkins syndrome, an autism-spectrum disorder characterized by developmental delay. Several disease-associated missense mutations in TCF4 have been shown to interfere with TCF4 function, but for many mutations, the impact remains undefined. Here, we tested the effects of 12 functionally uncharacterized disease-associated missense mutations and variations in TCF4 using transient expression in mammalian cells, confocal imaging, in vitro DNA-binding assays, and reporter assays. We show that Pitt-Hopkins syndrome-associated missense mutations within the basic helix-loop-helix domain of TCF4 and a Rett-like syndrome-associated mutation in a transcription activation domain result in altered DNA-binding and transcriptional activity of the protein. Some of the missense variations found in schizophrenia patients slightly increase TCF4 transcriptional activity, whereas no effects were detected for missense mutations linked to mild intellectual disability. We in addition find that the outcomes of several disease-related mutations are affected by cell type, TCF4 isoform, and dimerization partner, suggesting that the effects of TCF4 mutations are context-dependent. Together with previous work, this study provides a basis for the interpretation of the functional consequences of TCF4 missense variants.

EKLF/Klf1 Regulates Erythroid Transcription By Its Pioneering Activity and Subsequent Control of RNA Pol II Pause-Release

EKLF/Klf1 is a master transcriptional activator of critical genes that regulate both erythroid fate specification and terminal erythroid maturation. EKLF binds to DNA using three Zn-fingers at its C-terminus while the N-terminus constitutes a transcription activation domain (TAD) that interacts with various transcription co-factors including the protein acetylase CBP. An autosomal semi-dominant mutation at a single residue (E339D) in the mouse EKLF Zn-finger leads to Neonatal anemia (Nan). A mutation at the same residue in human EKLF (E325K) causes Congenital Dyserythropoietic Anemia type IV (CDA IV). Nan/Nan mice show lethality at embryonic day E10-11, in contrast to EKLF-/- homozygotes that survive until E15. Nan/+ heterozygotes survive to adulthood but are severely anemic, unlike EKLF+/- heterozygotes that display no aberrant phenotypes. The Nan-EKLF protein has an altered DNA binding specificity leading to a vastly altered transcriptome by two mechanisms. First, Nan-EKLF binding causes ectopic gene expression that significantly contributes to the severe anemia in Nan/+. Second, a subset of EKLF targets is downregulated in heterozygous Nan/+ mutants despite the presence of one copy of wild type EKLF, exacerbating the anemia. Thus, uncovering the mechanism by which gene expression is altered in Nan/+ may illuminate how EKLF normally activates transcription of its targets in vivo.To this end, we first examined the global occupancy of RNA Pol II phospho-Ser5 (as a paused mark) and phospho-Ser2 (as an elongation mark) in the mouse E13.5 fetal liver as a source of primary definitive erythroid cells. At promoters of ectopically expressed genes, where only Nan-EKLF (but not WT) binding is expected, we predominantly find increased levels of both paused and elongating RNA Pol II suggesting that Nan-EKLF binding activates transcription at ectopic genes by RNA Pol II recruitment and promoter proximal pausing. Further, we find increased levels of H3K27ac and CBP occupancy at these sites indicating that the mechanism of Pol II recruitment relies on CBP-mediated H3K27 acetylation and increased chromatin accessibility. Overall, this suggests robust pioneering activity of Nan-EKLF likely mediated by the interaction of its TAD with the CBP/p300 acetylase complex.At genes downregulated in Nan/+ we find two major patterns of Pol II occupancy. One is the converse of that seen at ectopic genes wherein there is a concomitant decrease in both Pol II p-Ser5 and p-Ser2 levels, along with lower H3K27ac and CBP levels suggesting EKLF gene activation has been lost at these sites in Nan/+. This includes cell cycle EKLF targets such as E2f2 and Rgcc. The second set of genes have comparable levels of p-Ser5 (paused) Pol II in Nan/+ and WT, but lower levels of p-Ser2 (elongating) Pol II in Nan/+. This suggests that although Pol II is being recruited to the TSS and pauses effectively, the pause-release step leading to effective transcription elongation is impaired. This subset includes important EKLF targets such as Bcl11a, Pax7, Xpo7, and several membrane transporters. As expected, CBP and H3K27ac levels are similar in WT and Nan/+ at these sites.To determine the cause of impaired RNA Pol II pause-release we examined the global occupancies of key transcription elongation factors such as P-TEFb and NELF. We find that levels of NELF, a negative elongation factor, remain unchanged in WT and Nan/+. However, levels of the P-TEFb subunit Cdk9, a positive elongation factor that facilitates release of paused RNA Pol II, is significantly lower at the TSS of these genes in Nan/+. This suggests that in Nan/+, possible reduction or loss of EKLF binding at some EKLF target promoters impairs effective recruitment of positive transcription elongation factors, resulting in a failure to efficiently release paused RNA Pol II. This causes downregulation of these EKLF target genes and contributes to the severe anemic phenotypes of the Nan mouse.We conclude that: EKLF exhibits expression control of its target genes at both the transcriptional initiation and elongation steps in vivo; EKLF can act as a pioneer transcription factor and increase chromatin accessibility through H3K27 acetylation by CBP leading to recruitment and pausing of RNA Pol II; and EKLF recruits the positive transcription elongation complex P-TEFb, enabling the controlled release of paused RNA Pol II at transcription start sites of a select group of its targets. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Dendritic Synaptic Morphology and Transcription by the SRF Cofactor MKL/MRTF.

Accumulating evidence suggests that the serum response factor (SRF) cofactor megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF) has critical roles in many physiological and pathological processes in various cell types. MKL/MRTF molecules comprise MKL1/MRTFA and MKL2/MRTFB, which possess actin-binding motifs at the N-terminus, and SRF-binding domains and a transcriptional activation domain (TAD) at the C-terminus. Several studies have reported that, in association with actin rearrangement, MKL/MRTF translocates from the cytoplasm to the nucleus, where it regulates SRF-mediated gene expression and controls cell motility. Therefore, it is important to elucidate the roles of MKL/MRTF in the nervous system with regard to its structural and functional regulation by extracellular stimuli. We demonstrated that MKL/MRTF is highly expressed in the brain, especially the synapses, and is involved in dendritic complexity and dendritic spine maturation. In addition to the positive regulation of dendritic complexity, we identified several MKL/MRTF isoforms that negatively regulate dendritic complexity in cortical neurons. We found that the MKL/MRTF isoforms were expressed differentially during brain development and the impacts of these isoforms on the immediate early genes including Arc/Arg3.1, were different. Here, we review the roles of MKL/MRTF in the nervous system, with a special focus on the MKL/MRTF-mediated fine-tuning of neuronal morphology and gene transcription. In the concluding remarks, we briefly discuss the future perspectives and the possible involvement of MKL/MRTF in neurological disorders such as schizophrenia and autism spectrum disorder.

Inactivation of the CIC-DUX4 oncogene through P300/CBP inhibition, a therapeutic approach for CIC-DUX4 sarcoma

CIC-DUX4 sarcoma (CDS) is a highly aggressive and metastatic small round type of predominantly pediatric sarcoma driven by a fusion oncoprotein comprising the transcriptional repressor Capicua (CIC) fused to the C-terminal transcriptional activation domain of DUX4. CDS rapidly develops resistance to chemotherapy, thus novel specific therapies are greatly needed. We demonstrate that CIC-DUX4 requires P300/CBP to induce histone H3 acetylation, activate its targets, and drive oncogenesis. We describe the synthetic route to a selective and highly potent P300/CBP inhibitor named iP300w and related stereoisomers, and find that iP300w efficiently suppresses CIC-DUX4 transcriptional activity and reverses CIC-DUX4 induced acetylation. iP300w is active at 100-fold lower concentrations than related stereoisomers or A-485. At low doses, iP300w shows specificity to CDS cancer cell lines, rapidly inducing cell cycle arrest and preventing growth of established CDS xenograft tumors when delivered in vivo. The effectiveness of iP300w to inactivate CIC-DUX4 highlights a promising therapeutic opportunity for CDS.

Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B

Photoactivated phytochrome B (PHYB) binds to antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to regulate hundreds of light responsive genes in Arabidopsis by promoting PIF degradation. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (AD) consisting of a hydrophobic activator motif flanked by acidic residues. A PIF3mAD mutant, in which the activator motif is replaced with alanines, fails to activate PIF3 target genes in Arabidopsis, validating the functions of the PIF3 AD in vivo. Intriguingly, the N-terminal photosensory module of PHYB binds immediately adjacent to the PIF3 AD to repress PIF3’s transactivation activity, demonstrating a novel PHYB signaling mechanism through direct interference of the transactivation activity of PIF3. Our findings indicate that PHYB, likely also PHYA, controls the stability and activity of PIFs via structurally separable dual signaling mechanisms.

Functional analysis of the transcriptional activator XlnR of Penicillium oxalicum.

The aim of this article is to study the functional features of Penicillium oxalicum transcriptional activator XlnR. The yeast reporter system was used to identify transcriptional activation domain of XlnR in P. oxalicum. The expression cassette was introduced into the xlnR locus of P. oxalicum by homologous recombination. In this study, several putative structural domains in P. oxalicum XlnR were predicted by bioinformatics analysis, and the transcriptional activation domain (351-694 region) was identified in XlnR relying on reporter gene system in yeast. In addition, the amino acid at XlnR 871 site (alanine) located in the regulatory region could influence the regulatory activity of XlnR directly. When the alanine at XlnR 871 site was replaced by stronger hydrophobic amino acid (e.g. valine or isoleucine), the regulatory activity will be greatly improved, especially for the regulation of hemicellulase genes expression. When alanine at XlnR 871 site was mutated to a hydrophilic amino acid (e.g. aspartic acid or arginine), the regulatory activity of XlnR will be reduced. The 351-694 region of P. oxalicum XlnR was identified as transcriptional activation domain, and the regulatory activity of XlnR was greatly influenced by hydrophobicity of amino acid at 871 site of XlnR in P. oxalicum. The results will provide an effective target site to regulate the activity of XlnR and improve cellulase production of P. oxalicum.

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 used as a gene activation system exploiting its ability in recruiting various transcriptional activation domains (TADs) for enhancing expression of the target gene(s). Drought tolerant and resource-efficient crops like millets have the potential for mitigating effects of climate change and for enhancing food security. Objective: This study aimed to use the Setaria italica (foxtail millet) genome sequence in the identification of a target gene and the subsequent generation of sgRNAs for use in CRISPRa for conferring water logging 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 water logging tolerance. The study provides a step-by-step example for using publicly accessible databases and bioinformatics tools from NCBI and Phytozome for the identification and characterization of the ortholog from Setaria italica. Soft berry 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 useful example of how to advance in genomics research including the revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species, through the utilization of the available genome sequence.