Consensus Sites Research Articles

Evolutionary conservation of sequence motifs at sites of protein modification

Gene duplications are common in biology and are likely to be an important source of functional diversification and specialization. The yeast Saccharomyces cerevisiae underwent a whole-genome duplication event early in evolution, and a substantial number of duplicated genes have been retained. We identified more than 3500 instances where only one of two paralogous proteins undergoes posttranslational modification despite having retained the same amino acid residue in both. We also developed a web-based search algorithm (CoSMoS.c.) that scores conservation of amino acid sequences based on 1011 wild and domesticated yeast isolates and used it to compare differentially modified pairs of paralogous proteins. We found that the most common modifications-phosphorylation, ubiquitylation, and acylation but not N-glycosylation-occur in regions of high sequence conservation. Such conservation is evident even for ubiquitylation and succinylation, where there is no established 'consensus site' for modification. Differences in phosphorylation were not associated with predicted secondary structure or solvent accessibility but did mirror known differences in kinase-substrate interactions. Thus, differences in posttranslational modification likely result from differences in adjoining amino acids and their interactions with modifying enzymes. By integrating data from large-scale proteomics and genomics analysis, in a system with such substantial genetic diversity, we obtained a more comprehensive understanding of the functional basis for genetic redundancies that have persisted for 100 million years.

UV irradiation remodels the specificity landscape of transcription factors

Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hypermutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. Weapplied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all the TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs, we identified a surprising but reproducible effect at certain nonconsensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and nonconsensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.

Ribosomes in RNA Granules Are Stalled on mRNA Sequences That Are Consensus Sites for FMRP Association.

Local translation in neurons is partly mediated by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the granule fraction, defined as the pellet of sucrose gradients used to separate polysomes from monosomes. The mechanism of how elongating ribosomes are reversibly stalled and unstalled on mRNAs is still unclear. In the present study, we characterize the ribosomes in the granule fraction using immunoblotting, cryogenic electron microscopy (cryo-EM), and ribosome profiling. We find that this fraction, isolated from 5-d-old rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homologue. Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (1) an enrichment for footprint reads of mRNAs that interact with FMRPs and are associated with stalled polysomes, (2) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development, and (3) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared with those usually found in ribosome profiling studies, the footprint reads were longer and were mapped to reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the granule fraction to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall ribosomes during translation elongation in neurons.SIGNIFICANCE STATEMENT Neurons send mRNAs to synapses in RNA granules, where they are not translated until an appropriate stimulus is given. Here, we characterize a granule fraction obtained from sucrose gradients and show that polysomes in this fraction are stalled on consensus sequences in a specific state of translational arrest with extended ribosome-protected fragments. This finding greatly increases our understanding of how neurons use specialized mechanisms to regulate translation and suggests that many studies on neuronal translation may need to be re-evaluated to include the large fraction of neuronal polysomes found in the pellet of sucrose gradients used to isolate polysomes.

Insight into the intrinsically disordered transactivation domain of NFkB as a regulator of DNA binding using UV-induced unnatural amino acid crosslinking.

Within our vast genome, specific transcription factors like NFkB manage to find target sequences amongst overwhelming nonspecific options and help elicit timely cellular responses. A transcription factor's binding affinity for specific vs. nonspecific DNA must therefore be in a delicate balance to facilitate this search. Key in vitro experiments that described these binding affinities for the dominant NFkB dimer RelA/p50 were carried out with a truncated version of RelA - excluding a large intrinsically disordered transactivation domain (TAD) that was believed to be important for transcription activation through cofactor interactions but not impactful for DNA binding. This fit with a traditional view of protein domains acting as modular units. More recently, however, we discovered a novel function of the TAD: binding experiments revealed that the TAD's presence increased RelA/p50's affinity for DNA, with the strongest increase in binding affinity observed for non-specific DNA sequences lacking kB consensus sites. Here we follow up these findings with an investigation of the TAD's mechanism of action. We present preliminary results using an unnatural amino acid crosslinker (p-benzoyl-l-phenylalanine or pBpF) in combination with mass spectrometry to probe TAD interactions that lead to the observed changes in DNA binding affinity and specificity. pBpF is incorporated into specific positions in the TAD using AMBER codon suppression, and crosslinking is initiated by UV light. Interactions are analyzed by mass spectrometry to identify key sites of contact between the TAD and the natively folded Rel-Homology Domain.

The C-terminus of the cargo receptor Erv14 affects COPII vesicle formation and cargo delivery.

The endoplasmic reticulum (ER) is the start site of the secretory pathway, where newly synthesized secreted and membrane proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Little is known about how post-translational modification events regulate packaging of cargo into COPII vesicles. The Saccharomyces cerevisiae protein Erv14, also known as cornichon, belongs to a conserved family of cargo receptors required for the selection and ER export of transmembrane proteins. In this work, we show the importance of a phosphorylation consensus site (S134) at the C-terminus of Erv14. Mimicking phosphorylation of S134 (S134D) prevents the incorporation of Erv14 into COPII vesicles, delays cell growth, exacerbates growth of sec mutants, modifies ER structure and affects localization of several plasma membrane transporters. In contrast, the dephosphorylated mimic (S134A) had less deleterious effects, but still modifies ER structure and slows cell growth. Our results suggest that a possible cycle of phosphorylation and dephosphorylation is important for the correct functioning of Erv14.

Neuroprotection by Skimmianine in Lipopolysaccharide-Activated BV-2 Microglia.

Skimmianine is a furoquinoline alkaloid which is found in the Zanthoxylum genus and also in other plants of the Rutaceae family. This study evaluated the effects of skimmianine on the production of pro-inflammatory mediators in LPS-activated BV-2 microglia. Cultured BV-2 cells were treated with skimmianine (10, 20 and 30 μM), followed by stimulation with LPS (100 ng/mL). Levels of TNFα and IL-6 in cell supernatants were measured using ELISA, while NO and PGE2 levels were evaluated with Griess assay and EIA, respectively. Western blotting was used to determine the protein expression of iNOS, COX-2, phospho-p65 and phospho-IκBα. Results showed that Skimmianine reduced LPS-induced elevated the secretion of TNFα, IL-6, NO, and PGE2, as well as the increased protein expression of iNOS and COX-2. Experiments to elucidate the mechanisms of the anti-neuroinflammatory activity of skimmianine revealed the significant inhibition of LPS-induced increased NF-κB-mediated luciferase activity. Pre-treatment with skimmianine also reduced LPS-induced the increased phosphorylation of NF-κB/p65 and IκBα proteins. Furthermore, skimmianine interfered with the binding capacity of NF-κB to consensus sites. Skimmianine pre-treatment protected HT-22 cells from toxicity induced by microglia-conditioned media, as well as increasing MAP-2 expression. The results of this study suggest that skimmianine inhibits neuroinflammation in LPS-activated microglia by targeting the NF-κB activation pathway. Skimmianine also produced neuroprotection against neurotoxicity induced by microglia-conditioned media.

Real-time observation of functional specialization among phosphorylation sites in CFTR.

Phosphoregulation is ubiquitous in biology. Defining the functional roles of individual phosphorylation sites within a multivalent system remains particularly challenging. We have therefore applied a chemical biology approach to light-control the state of single candidate phosphoserines in the canonical anion channel CFTR while simultaneously measuring channel activity. The data show striking non-equivalency among protein kinase A consensus sites, which vary from <10% to >1,000% changes in channel activity upon phosphorylation. Of note, slow phosphorylation of S813 suggests that this site is rate-limiting to the full activation of CFTR. Further, this approach reveals an unexpected coupling between the phosphorylation of S813 and a nearby site, S795. Overall, these data establish an experimental route to understanding roles of specific phosphoserines within complex phosphoregulatory domains. This strategy may be employed in the study of phosphoregulation of other eukaryotic proteins.

MiT/TFE Family Renal Cell Carcinoma.

The microphthalmia-associated transcription factor/transcription factor E (MiT/TFE) family of transcription factors are evolutionarily conserved, basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors, consisting of MITF, TFEB, TFE3, and TFEC. MiT/TFE proteins, with the exception of TFEC, are involved in the development of renal cell carcinoma (RCC). Most of the MiT/TFE transcription factor alterations seen in sporadic RCC cases of MiT family translocation renal cell carcinoma (tRCC) are chimeric proteins generated by chromosomal rearrangements. These chimeric MiT/TFE proteins retain the bHLH-Zip structures and act as oncogenic transcription factors. The germline variant of MITF p.E318K has been reported as a risk factor for RCC. E 318 is present at the SUMOylation consensus site of MITF. The p.E318K variant abrogates SUMOylation on K 316, which results in alteration of MITF transcriptional activity. Only a few cases of MITF p.E318K RCC have been reported, and their clinical features have not yet been fully described. It would be important for clinicians to recognize MITF p.E318K RCC and consider MITF germline testing for undiagnosed familial RCC cases. This review outlines the involvement of the MiT/TFE transcription factors in RCC, both in sporadic and hereditary cases. Further elucidation of the molecular function of the MiT/TFE family is necessary for better diagnosis and treatment of these rare diseases.

Assaying for Arginyltransferase Activity and Specificity by Peptide Arrays.

Here, we describe arginylation assays performed on peptide arrays immobilized on cellulose membranes via chemical synthesis. In this assay, it is possible to simultaneously compare arginylation activity on hundreds of peptide substrates to analyze the specificity of arginyltransferase ATE1 toward its target site(s) and the amino acid sequence context. This assay was successfully employed in prior studies to dissect the arginylation consensus site and enable predictions of arginylated proteins encoded in eukaryotic genomes.

FOXO3a Mediates Homologous Recombination Repair (HRR) via Transcriptional Activation of MRE11, BRCA1, BRIP1, and RAD50

To test whether homologous recombination repair (HRR) depends on FOXO3a, a cellular aging model of human dermal fibroblast (HDF) and tet-on flag-h-FOXO3a transgenic mice were studied. HDF cells transfected with over-expression of wt-h-FOXO3a increased the protein levels of MRE11, BRCA1, BRIP1, and RAD50, while knock-down with siFOXO3a decreased them. The protein levels of MRE11, BRCA1, BRIP1, RAD50, and RAD51 decreased during cellular aging. Chromatin immunoprecipitation (ChIP) assay was performed on FOXO3a binding accessibility to FOXO consensus sites in human MRE11, BRCA1, BRIP1, and RAD50 promoters; the results showed FOXO3a binding decreased during cellular aging. When the tet-on flag-h-FOXO3a mice were administered doxycycline orally, the protein and mRNA levels of flag-h-FOXO3a, MRE11, BRCA1, BRIP1, and RAD50 increased in a doxycycline-dose-dependent manner. In vitro HRR assays were performed by transfection with an HR vector and I-SceI vector. The mRNA levels of the recombined GFP increased after doxycycline treatment in MEF but not in wt-MEF, and increased in young HDF comparing to old HDF, indicating that FOXO3a activates HRR. Overall, these results demonstrate that MRE11, BRCA1, BRIP1, and RAD50 are transcriptional target genes for FOXO3a, and HRR activity is increased via transcriptional activation of MRE11, BRCA1, BRIP1, and RAD50 by FOXO3a.

Mechano-growth factor E-domain modulates cardiac contractile function through 14-3-3 protein interactomes.

In the heart, alternative splicing of the igf-I gene produces two isoforms: IGF-IEa and IGF-IEc, (Mechano-growth factor, MGF). The sequence divergence between their E-domain regions suggests differential isoform function. To define the biological actions of MGF's E-domain, we performed in silico analysis of the unique C-terminal sequence and identified a phosphorylation consensus site residing within a putative 14-3-3 binding motif. To test the functional significance of Ser 18 phosphorylation, phospho-mimetic (S/E18) and phospho-null (S/A18) peptides were delivered to mice at different doses for 2 weeks. Cardiovascular function was measured using echocardiography and a pressure-volume catheter. At the lowest (2.25mg/kg/day) and highest (9mg/kg/day) doses, the peptides produced a depression in systolic and diastolic parameters. However, at 4.5mg/kg/day the peptides produced opposing effects on cardiac function. Fractional shortening analysis also showed a similar trend, but with no significant change in cardiac geometry. Microarray analysis discovered 21 genes (FDR p < 0.01), that were expressed accordant with the opposing effects on contractile function at 4.5mg/kg/day, with the nuclear receptor subfamily 4 group A member 2 (Nr4a2) identified as a potential target of peptide regulation. Testing the regulation of the Nr4a family, showed the E-domain peptides modulate Nr4a gene expression following membrane depolarization with KCl in vitro. To determine the potential role of 14-3-3 proteins, we examined 14-3-3 isoform expression and distribution. 14-3-3γ localized to the myofilaments in neonatal cardiac myocytes, the cardiac myocytes and myofilament extracts from the adult heart. Thermal shift analysis of recombinant 14-3-3γ protein showed the S/A18 peptide destabilized 14-3-3γ folding. Also, the S/A18 peptide significantly inhibited 14-3-3γ's ability to interact with myosin binding protein C (MYPC3) and phospholamban (PLN) in heart lysates from dobutamine injected mice. Conversely, the S/E18 peptide showed no effect on 14-3-3γ stability, did not inhibit 14-3-3γ's interaction with PLN but did inhibit the interaction with MYPC3. Replacing the glutamic acid with a phosphate group on Ser 18 (pSer18), significantly increased 14-3-3γ protein stability. We conclude that the state of Ser 18 phosphorylation within the 14-3-3 binding motif of MGF's E-domain, modulates protein-protein interactions within the 14-3-3γ interactome, which includes proteins involved in the regulation of contractile function.

Transcriptional Regulation, Signaling Pathways, and Subcellular Localization of Corticotropin-Releasing Factor Receptors in the Central Nervous System.

Corticotropin-releasing factor (CRF) receptors CRF-R1 and CRF-R2 are differentially distributed in body tissues, and although they respond differentially to stimuli due to their association with different signaling pathways, both receptors have a fundamental role in the response and adaptation to stressful stimuli. Here, we summarize the reported data on different forms of CRF-R1 and CRF-R2 regulation as well as on their subcellular localization. Although the presence of R1 has been described at pre- and postsynaptic sites, R2 is mainly associated with postsynaptic densities. Different studies have provided valuable information on how these receptors regulate responses at a central level, elucidating different and sometimes synergistic roles in response to stress, but despite their high sequence identity, both receptors have been described to be differentially regulated both by their ligands and by transcriptional factors. To date, and from the point of view of their promoter sequences, it has not yet been reported how the different consensus sites identified in silico could be modulating the transcriptional regulation and expression of the receptors under different conditions, which strongly limits the full understanding of their differential functions, providing a wide field to increase and expand the study of the regulation and role of CRF receptors in the CRF system. SIGNIFICANCE STATEMENT: A large number of physiological functions related to the organization of the stress response in different body tissues are associated with the corticotropin-releasing factor system. This system also plays a relevant role in depression and anxiety disorders, as well as being a direct connection between stress and addiction. A better understanding of how the receptors of this system are regulated would help to expand the understanding of how these receptors respond differently to both drugs and stressful stimuli.

Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance

Binding to binding site clusters has yet to be characterized in depth, and the functional relevance of low-affinity clusters remains uncertain. We characterized transcription factor binding to low-affinity clusters in vitro and found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, small clusters with binding sites an order of magnitude lower in affinity give rise to high mean occupancies at physiologically-relevant transcription factor concentrations. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters in a synthetic and native gene regulatory network in S. cerevisiae. In both systems, clusters of low-affinity binding sites generated transcriptional output comparable to single or even multiple consensus sites. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.

Salt Dependence of DNA Binding Activity of Human Transcription Factor Dlx3.

Distal-less 3 (Dlx3) is a homeobox-containing transcription factor and plays a crucial role in the development and differentiation process. Human Dlx3 consists of two transactivation domains and a homeobox domain (HD) that selectively binds to the consensus site (5′-TAATT-3′) of the DNA duplex. Here, we performed chemical shift perturbation experiments on Dlx3-HD in a complex with a 10-base-paired (10-bp) DNA duplex under various salt conditions. We also acquired the imino proton spectra of the 10-bp DNA to monitor the changes in base-pair stabilities during titration with Dlx3-HD. Our study demonstrates that Dlx3-HD selectively recognizes its consensus DNA sequences through the α3 helix and L1 loop regions with a unique dynamic feature. The dynamic properties of the binding of Dlx3-HD to its consensus DNA sequence can be modulated by varying the salt concentrations. Our study suggested that this unique structural and dynamic feature of Dlx3-HD plays an important role in target DNA recognition, which might be associated with tricho-dento-osseous syndrome.

Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana.

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

Engineering of a nitrilase through consensus sequence analysis and conserved site substitution to improve its thermostability and activity

The thermostability of nitrilase from different microorganisms varies widely and is closely related to conserved sequences. In this study, a semi-rational design based on sequence analysis was performed to improve the thermostability and specific activity of the regioselective nitrilase from Acidovorax facilis ZJB09122. Using thermostable nitrilases from thermophilic microorganisms as templates, consensus sites 151, 223, 250, 169 and 280 were found to have effects on the thermostability and enzyme activity. A triple mutant AcN-T151V/C223A/C250G (AcN-T) was obtained with 15.9% higher activity and 2.1-fold longer half-life at 50 °C compared with the parent AcN-M. The optimal reaction temperature was 55 °C and the product tolerance was also improved. It was found that site 250 close to the catalytic active center plays a key role in the thermostability of nitrilase, and this role is universal among homologous nitrilases. On the other hand, sites 151 and 223 mainly affect enzyme activity, and this role is not universal.

A Test and Refinement of Folding Free Energy Nearest Neighbor Parameters for RNA Including N6-Methyladenosine

RNA folding free energy change parameters are widely used to predict RNA secondary structure and to design RNA sequences. These parameters include terms for the folding free energies of helices and loops. Although the full set of parameters has only been traditionally available for the four common bases and backbone, it is well known that covalent modifications of nucleotides are widespread in natural RNAs. Covalent modifications are also widely used in engineered sequences. We recently derived a full set of nearest neighbor terms for RNA that includes N6-methyladenosine (m6A). In this work, we test the model using 98 optical melting experiments, matching duplexes with or without N6-methylation of A. Most experiments place RRACH, the consensus site of N6-methylation, in a variety of contexts, including helices, bulge loops, internal loops, dangling ends, and terminal mismatches. For matched sets of experiments that include either A or m6A in the same context, we find that the parameters for m6A are as accurate as those for A. Across all experiments, the root mean squared deviation between estimated and experimental free energy changes is 0.67 kcal/mol. We used the new experimental data to refine the set of nearest neighbor parameter terms for m6A. These parameters enable prediction of RNA secondary structures including m6A, which can be used to model how N6-methylation of A affects RNA structure.

In Silico Insights towards the Identification of SARS-CoV-2 NSP13 Helicase Druggable Pockets.

The merging of distinct computational approaches has become a powerful strategy for discovering new biologically active compounds. By using molecular modeling, significant efforts have recently resulted in the development of new molecules, demonstrating high efficiency in reducing the replication of severe acute respiratory coronavirus 2 (SARS-CoV-2), the agent responsible for the COVID-19 pandemic. We have focused our interest on non-structural protein Nsp13 (NTPase/helicase), as a crucial protein, embedded in the replication–transcription complex (RTC), that controls the virus life cycle. To assist in the identification of the most druggable surfaces of Nsps13, we applied a combination of four computational tools: FTMap, SiteMap, Fpocket and LigandScout. These software packages explored the binding sites for different three-dimensional structures of RTC complexes (PDB codes: 6XEZ, 7CXM, 7CXN), thus, detecting several hot spots, that were clustered to obtain ensemble consensus sites, through a combination of four different approaches. The comparison of data provided new insights about putative druggable sites that might be employed for further docking simulations on druggable surfaces of Nsps13, in a scenario of repurposing drugs.

Angiopathic activity of LRG1 is induced by the IL-6/STAT3 pathway

Leucine-rich α-2-glycoprotein 1 (LRG1) is a secreted glycoprotein that under physiological conditions is produced predominantly by the liver. In disease, its local induction promotes pathogenic neovascularisation while its inhibition leads to reduced dysfunctional angiogenesis. Here we examine the role of interleukin-6 (IL-6) in defective angiogenesis mediated by LRG1. IL-6 treatment induced LRG1 expression in endothelial cells and ex vivo angiogenesis cultures and promoted vascular growth with reduced mural cell coverage. In Lrg1−/− explants, however, IL-6 failed to stimulate angiogenesis and vessels exhibited improved mural cell coverage. IL-6 activated LRG1 transcription through the phosphorylation and binding of STAT3 to a conserved consensus site in the LRG1 promoter, the deletion of which abolished activation. Blocking IL-6 signalling in human lung endothelial cells, using the anti-IL6 receptor antibody Tocilizumab, significantly reduced LRG1 expression. Our data demonstrate that IL-6, through STAT3 phosphorylation, activates LRG1 transcription resulting in vascular destabilisation. This observation is especially timely in light of the potential role of IL-6 in COVID-19 patients with severe pulmonary microvascular complications, where targeting IL-6 has been beneficial. However, our data suggest that a therapy directed towards blocking the downstream angiopathic effector molecule LRG1 may be of greater utility.

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA.

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.