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Directed evolution of proteins is critical for applications in basic biological research, therapeutics, diagnostics, and sustainability. However, directed evolution methods are labor intensive, cannot efficiently optimize over multiple protein properties, and are often trapped by local maxima. In silico-directed evolution methods incorporating protein language models (PLMs) have the potential to accelerate this engineering process, but current approaches fail to generalize across diverse protein families. We introduce EVOLVEpro, a few-shot active learning framework to rapidly improve protein activity using a combination of PLMs and protein activity predictors, achieving improved activity with as few as four rounds of evolution. EVOLVEpro substantially enhances the efficiency and effectiveness of in silico protein evolution, surpassing current state-of-the-art methods and yielding proteins with up to 100-fold improvement of desired properties. We showcase EVOLVEpro for five proteins across three applications: T7 RNA polymerase for RNA production, a miniature CRISPR nuclease, a prime editor, and an integrase for genome editing, and a monoclonal antibody for epitope binding. These results demonstrate the advantages of few-shot active learning with small amounts of experimental data over zero-shot predictions. EVOLVEpro paves the way for broader applications of AI-guided protein engineering in biology and medicine.
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RNA editing offers the opportunity to introduce either stable or transient modifications to nucleic acid sequence without permanent off-target effects, but installation of arbitrary edits into the transcriptome is currently infeasible. Here, we describe Programmable RNA Editing & Cleavage for Insertion, Substitution, and Erasure (PRECISE), a versatile RNA editing method for writing RNA of arbitrary length and sequence into existing pre-mRNAs via 5' or 3' trans-splicing. In trans-splicing, an exogenous template is introduced to compete with the endogenous pre-mRNA, allowing for replacement of upstream or downstream exon sequence. Using Cas7-11 cleavage of pre-mRNAs to bias towards editing outcomes, we boost the efficiency of RNA trans-splicing by 10-100 fold, achieving editing rates between 5-50% and 85% on endogenous and reporter transcripts, respectively, while maintaining high-fidelity. We demonstrate PRECISE editing across 11 distinct endogenous transcripts of widely varying expression levels, showcasing more than 50 types of edits, including all 12 possible transversions and transitions, insertions ranging from 1 to 1,863 nucleotides, and deletions. We show high efficiency replacement of exon 4 of MECP2, addressing most mutations that drive the Rett Syndrome; editing of SHANK3 transcripts, a gene involved in Autism; and replacement of exon 1 of HTT, removing the hallmark repeat expansions of Huntington's disease. Whole transcriptome sequencing reveals the high precision of PRECISE editing and lack of off-target trans-splicing activity. Furthermore, we combine payload engineering and ribozymes for protein-free, high-efficiency trans-splicing, with demonstrated efficiency in editing HTT exon 1 via AAV delivery. We show that the high activity of PRECISE editing enables editing in non-dividing neurons and patient-derived Huntington's disease fibroblasts. PRECISE editing markedly broadens the scope of genetic editing, is straightforward to deliver over existing gene editing tools like prime editing, lacks permanent off-targets, and can enable any type of genetic edit large or small, including edits not otherwise possible with existing RNA base editors, widening the spectrum of addressable diseases.
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Our ability to edit genomes lags behind our capacity to sequence them, but the growing understanding of CRISPR biology and its application to genome, epigenome and transcriptome engineering is narrowing this gap. In this Review, we discuss recent developments of various CRISPR-based systems that can transiently or permanently modify the genome and the transcriptome. The discovery of further CRISPR enzymes and systems through functional metagenomics has meaningfully broadened the applicability of CRISPR-based editing. Engineered Cas variants offer diverse capabilities such as base editing, prime editing, gene insertion and gene regulation, thereby providing a panoply of tools for the scientific community. We highlight the strengths and weaknesses of current CRISPR tools, considering their efficiency, precision, specificity, reliance on cellular DNA repair mechanisms and their applications in both fundamental biology and therapeutics. Finally, we discuss ongoing clinical trials that illustrate the potential impact of CRISPR systems on human health.
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Sistemas CRISPR-Cas , Epigenoma , Edición Génica , Transcriptoma , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Epigenoma/genética , Animales , Transcriptoma/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genoma/genéticaRESUMEN
BACKGROUND: Women with multiple pregnancies often experience abdominal protrusion and/or a lax abdominal wall. Various open surgical techniques have been developed to address rectus diastasis in abdominoplasty, ranging from suture plication to mesh reinforcement. This study aims to compare the clinical and radiological changes between traditional abdominal plication and the addition of non-absorbable mesh for rectus muscle (RM) diastasis repair in terms of function, postoperative outcome, and recurrence. PATIENTS AND METHOD: This prospective retrospective study involved 63 women who underwent cosmetic tummy tuck surgery and met certain eligibility criteria. Patients with only mild diastasis recti, midline hernia, contraindications for major surgery, recent smoking history, or refusal of mesh augmentation were excluded. Clinical examination for abdominal protrusion or bulging and CT imaging was performed to check for recurrence of diastasis recti. The study included 33 patients who underwent mesh repair and 30 who underwent traditional abdominal plication. Follow-up was conducted after 1 year using CT and a questionnaire to assess various factors compared to preoperative measurements, with overall satisfaction rated on a 10-point Likert scale. RESULTS: There was no significant difference in demographic data between the two groups. Patients who underwent mesh repair had a slightly longer hospital stay and drain duration. The average waist circumference decreased in both groups without any statistically significant difference. Objective CT showed significant reductions in both groups in inter-rectus distance, RM width and circumference, and intra-abdominal circumference compared to preoperative values. All patients expressed satisfaction with scar quality and umbilicus aesthetics, and no recurrence was detected either clinically or radiologically during the follow-up period. CONCLUSION: Comprehensive preoperative assessment and imaging techniques like ultrasound and CT scans allow surgeons to detect postpartum changes in the abdominal wall. Mesh reinforcement may be indicated for diastasis above 4 cm in obese multiparous females. Thorough preoperative evaluation permits customized surgical plans to optimally restore abdominal wall anatomy and function. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors at www.springer.com/00266 .
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Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.
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Eucariontes , Virus , Humanos , Eucariontes/genética , Eucariontes/metabolismo , Desoxirribonucleasa I , ARN/genética , Desoxirribonucleasas/metabolismo , Virus/genéticaRESUMEN
TnpB proteins are RNA-guided nucleases that are broadly associated with IS200/605 family transposons in prokaryotes. TnpB homologs, named Fanzors, have been detected in genomes of some eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. We searched genomes of diverse eukaryotes and their viruses for TnpB homologs and identified numerous putative RNA-guided nucleases that are often associated with various transposases, suggesting they are encoded in mobile genetic elements. Reconstruction of the evolution of these nucleases, which we rename Horizontally-transferred Eukaryotic RNA-guided Mobile Element Systems (HERMES), revealed multiple acquisitions of TnpBs by eukaryotes and subsequent diversification. In their adaptation and spread in eukaryotes, HERMES proteins acquired nuclear localization signals, and genes captured introns, indicating extensive, long term adaptation to functioning in eukaryotic cells. Biochemical and cellular evidence show that HERMES employ non-coding RNAs encoded adjacent to the nuclease for RNA-guided cleavage of double-stranded DNA. HERMES nucleases contain a re-arranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that HERMES can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.
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Klebsiella pneumoniae producing extended-spectrum ß-lactamases (ESBL) continues to pose huge therapeutic challenges in the treatment of infections, primarily urinary infections, due to its multidrug resistance to antibiotics. Therefore, there is a need for research on this topic to investigate ways to reduce the spread of antibiotic resistance, identify novel therapeutic approaches to treat these infections and gain a better understanding of the mechanisms of resistance. In this context, this study aimed to analyze the chemical composition of essential oils (EOs) of Thymus algeriensis, Syzygium aromaticum, and Eucalyptus globulus and assess their activity against K. pneumoniae ESBL strains, as well as the interaction type between these EOs and antibiotics used for the treatment of K. pneumoniae ESBL infections. The composition of the EOs was determined by gas chromatography-mass spectrometry (GC-MS). The activity of EOs was tested using the disc diffusion and liquid microdilution methods. The type of interaction between EOs and antibiotics was studied using the agar disk diffusion and chessboard methods. The analysis of the EO of T. algeriensis showed that the main compounds were thymol (23.14%), linalool (18.44%), and p-cymene (16.17%). The main constituents of EO of E. globulus were eucalyptol (54.29%), α-pinene (17.32%), aromadendrene (7.02%), and pinocarveol (6.32%). As for the EO of S. aromaticum, the major constituents were eugenol (80.46%) and eugenol acetate (16.23%). Results of the activity tests showed that all three EOs were active against the tested strains, with inhibition diameters ranging from 7.39±0.44mm to 32.4±1.05mm and minimum inhibitory concentrations (MICs) varying from 2 to 441.5±5.66 mg/ml. A synergistic interaction was obtained between amoxicillin-clavulanic acid and T. algeriensis EO against two strains of K. pneumoniae ESBL. These results demonstrate the potential of our EOs to inhibit multi-resistant pathogenic ESBL strains, as well as their synergistic interaction with antibiotics used in therapy, which could be an alternative to the use of antibiotics alone in treatment to fight against these multi-resistant pathogenic bacteria.
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Antibacterianos , Aceites Volátiles , Antibacterianos/farmacología , Aceites Volátiles/farmacología , Aceites Volátiles/química , Klebsiella pneumoniae , Eugenol , Timol , Bacterias , Pruebas de Sensibilidad MicrobianaRESUMEN
CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.
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Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Bacterias/genética , ARN Bacteriano/genética , ADNRESUMEN
Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.
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Diferenciación Celular , Factores de Transcripción , Humanos , Cromatina , Regulación de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Factores de Transcripción/metabolismo , Atlas como AsuntoRESUMEN
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.
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Sistemas CRISPR-Cas , Integrasas , Humanos , Sistemas CRISPR-Cas/genética , División del ADN , Edición Génica , ADN/genética , Reparación del ADN por Unión de Extremidades/genéticaRESUMEN
Programmable approaches to sense and respond to the presence of specific RNAs in biological systems have broad applications in research, diagnostics, and therapeutics. Here we engineer a programmable RNA-sensing technology, reprogrammable ADAR sensors (RADARS), which harnesses RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a cargo protein by the presence of endogenous RNA transcripts. Introduction of a stop codon in a guide upstream of the cargo makes translation contingent on binding of an endogenous transcript to the guide, leading to ADAR editing of the stop codon and allowing translational readthrough. Through systematic sensor engineering, we achieve 277 fold improvement in sensor activation and engineer RADARS with diverse cargo proteins, including luciferases, fluorescent proteins, recombinases, and caspases, enabling detection sensitivity on endogenous transcripts expressed at levels as low as 13 transcripts per million. We show that RADARS are functional as either expressed DNA or synthetic mRNA and with either exogenous or endogenous ADAR. We apply RADARS in multiple contexts, including tracking transcriptional states, RNA-sensing-induced cell death, cell-type identification, and control of synthetic mRNA translation.
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Proteínas de Unión al ARN , ARN , ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Codón de Terminación , Edición de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Methylene blue (MB) immobilized onto a sulfonated poly(glycidyl methacrylate) (SPGMA) polymer composite has been developed as a novel adsorbent for water treatment applications. The MB adsorptions onto sulfonated poly(glycidyl methacrylate) polymer characters have been studied. The adsorption isotherms, namely Langmuir and Freundlich, have been investigated. Other isotherm models. As a compromise between the Freundlich and Langmuir isotherm models, such as the D-R isotherm and the Temkin isotherm, have been compared. The results indicated that the adsorption process followed the Freundlich isotherm model, indicating heterogeneous surface site energies and multi-layer levels of sorption. This study selected three linear kinetic models, namely pseudo-first order, pseudo-second order, and Elovich, to describe the MB sorption process using SPGMA negatively charged nanoparticles (430 nm). The obtained data revealed that the adsorption process obeyed the pseudo-second-order kinetic model, suggesting that the rate-limiting step in these sorption processes may be chemisorption. Furthermore, the thermodynamic parameters have been evaluated. Moreover, the interaction of the MB molecules with SPGMA nanoparticles has been simulated using the governing equation that describes ion exchange resin derived from Nernst-Plank equations between two ion species. Finally, the developed MB-SPGMA composite adsorbent (27 mg/g) wastested for the first time for the removal of Cr6+ ions and Mn7+ metal ions from dichromate and permanganate-contaminated waters under mild adsorption conditions, opening a new field of multiuse of the same adsorbent in the removal of more than one contaminant.
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Contaminantes Químicos del Agua , Purificación del Agua , Azul de Metileno , Compuestos Azo , Polímeros , Concentración de Iones de Hidrógeno , Purificación del Agua/métodos , Cinética , Adsorción , Termodinámica , AlcanosulfonatosRESUMEN
In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.
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Proteínas Bacterianas , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Deltaproteobacteria , Endopeptidasas , Proteolisis , ARN Guía de Kinetoplastida , Humanos , Microscopía por Crioelectrón , Endopeptidasas/química , Endopeptidasas/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Deltaproteobacteria/enzimología , Deltaproteobacteria/genética , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/metabolismo , Factor sigma/metabolismo , Transcripción Genética , Especificidad por Sustrato , Regulación Alostérica , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Activación EnzimáticaRESUMEN
The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE (RNA polymerase, extracytoplasmic E), suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11-Csx29-Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11-Csx29 effector is an RNA-dependent nuclease-protease.
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Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Deltaproteobacteria , Endonucleasas , Proteolisis , ARN Guía de Kinetoplastida , Microscopía por Crioelectrón , Endonucleasas/química , Endonucleasas/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/metabolismo , Deltaproteobacteria/enzimología , Conformación Proteica , Células HEK293RESUMEN
Objective: The present clinical trial was conducted to evaluate the clinical performance of the biomimetic, bilayered structure utilizing a fiber reinforced bulk fill resin composite with a nanohybrid capping layer, compared to incremental packing of nanohybrid resin composite, in deep proximal cavities in permanent molars. Material and methods: A total of 36 deep proximal cavities in vital molars were restored either with a bilayered structure of fiber reinforced composite resin as a dentine substitute and a capping layer of nanohybrid composite resin (n=18) or conventional, nanohybrid composite resin incrementation (n=18). The restorations were assessed over a period of 12 months using the modified USPHS criteria. The criteria evaluated were: fracture and retention, marginal integrity, marginal discoloration, anatomic form, proximal contact, surface texture, radiographic evaluation, postoperative sensitivity and secondary caries. Results: There was no statistically or clinically significant difference between fiber-reinforced resin composite and conventional incremental resin composite. There was no risk for failure regarding all the evaluated modified USPHS criteria for both materials after 12 months (RR= 1(95% CI 0.0209 to 47.8503; P =1.0000)). Conclusion: The biomimetic approach utilizing a fiber reinforced resin composite dentine substitute showed a comparable clinical performance to nanohybrid resin composite incrementation. Bulk fill fiber reinforced resin composite is an efficient alternative in restoration of deep proximal cavities in posterior teeth. Further long-term studies are necessary to confirm these results.
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The paranasal sinuses are hollowed, air-filled cavities surrounding the nasal cavity. Many pathological processes affect the sinuses, but inflammatory conditions are the commonest, even in asymptomatic patients who undergo head imaging for other indications showing one or more abnormalities of the sinuses. Our research aims to determine the prevalence of incidental paranasal sinuses abnormalities seen among patients who undergo head CT scanning. In addition, it provides baseline information for further investigations required. The study was designed to evaluate all patients who underwent head CT scanning for any reason unrelated to paranasal sinuses abnormalities. 1849 cases were selected and retrospectively analyzed from the elective and emergency CT in the last nine months, from August 2020 to April 2021. In order to meet the inclusion criteria, indications for imaging must not be sinus-related. The study was conducted on 1849 cases who had undergone head CT scans for pathology, 1204 (65%) were male and 645 (35%) were female. Abnormalities of the sinuses were found in about 617 (33%) of all patients, with a higher rate in males (22.23%) than females (11.14%). In addition, these abnormalities were found in younger patients at a higher rate than in middle and old ages 19.74%, 7.19%, and 6.44%, respectively. Our findings revealed that the prevalence of paranasal sinuses abnormalities in asymptomatic Saudi patients was high (33%). Most of the affected sinuses were the maxillary. The male patients were more affected than females in all findings.
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Senos Paranasales , Adulto , Femenino , Humanos , Masculino , Cavidad Nasal , Senos Paranasales/diagnóstico por imagen , Senos Paranasales/patología , Estudios Retrospectivos , Arabia Saudita/epidemiología , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
PURPOSE: This study aims to investigate the relationship between OHRQoL and orofacial dysfunction in children practicing oral habits. METHODS: Thirty Egyptian Children, aged from five to seven years, practicing oral habits (habit practicing/exposed group), were examined for orofacial dysfunction using Nordic Orofacial Test-Screen (NOT-S). Their parents were asked to fill 8-item Parental-Caregiver Perception Questionnaire (P-CPQ), translated to Arabic, as an assessment tool for their children's OHRQoL. The scores of the habit practicing group were compared to those obtained from another 30 children with matched criteria not practicing oral habits (habit free/ control group). RESULTS: Children in the exposure group showed higher total NOT-S score (median 3, range 1-5) and higher P-CPQ (median 6, range 1-16) than the control group (median 0.5, range 0-2) and (median 4, range 1-8), with a statistical significance (p = 0.00, p = 0.014), respectively. A statistically significant moderate positive correlation was found between OHRQoL and orofacial dysfunction in the habit practicing group, (R = 0.384, p = 0.036). The exposure group was found to be 7.4 and 1.5 times the control group in developing orofacial dysfunction, and having inferior OHRQoL, respectively. CONCLUSION: An existing association between the degree of orofacial dysfunction and OHRQoL in children practicing oral habit(s) is suggested. TRIAL REGISTRATION NUMBER: NCT04575792, date of registration: 26/9/2020, first posted (approved): 5/10/2020.