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The genome-editing Cas9 protein uses multiple amino-acid residues to bind the target DNA. Considering only the residues in proximity to the target DNA as potential sites to optimise Cas9's activity, the number of combinatorial variants to screen through is too massive for a wet-lab experiment. Here we generate and cross-validate ten in silico and experimental datasets of multi-domain combinatorial mutagenesis libraries for Cas9 engineering, and demonstrate that a machine learning-coupled engineering approach reduces the experimental screening burden by as high as 95% while enriching top-performing variants by ∼7.5-fold in comparison to the null model. Using this approach and followed by structure-guided engineering, we identify the N888R/A889Q variant conferring increased editing activity on the protospacer adjacent motif-relaxed KKH variant of Cas9 nuclease from Staphylococcus aureus (KKH-SaCas9) and its derived base editor in human cells. Our work validates a readily applicable workflow to enable resource-efficient high-throughput engineering of genome editor's activity. Screening combinatorial mutants is too massive for wet-lab experiment alone. Here the authors present a machine learning-coupled combinatorial mutagenesis approach to vastly reduce experimental burden for engineering Cas9 genome editing enzymes. [ABSTRACT FROM AUTHOR]

Aim: To update the existing European Academy of Paediatric Dentistry (EAPD) 2010 policy document on the 'Best Clinical Practice guidance for clinicians dealing with children presenting with Molar-Incisor-Hypomineralisation (MIH).' Methods: Experts, assigned the EAPD, worked on two different topics: (A) Aetiological factors involved in MIH, and (B) Treatment options for the clinical management of MIH. The group prepared two detailed systematic reviews of the existing literature relevant to the topics and following a consensus process produced the updated EAPD policy document on the 'Best Clinical Practice guidance for clinicians dealing with children presenting with molar-incisor-hypomineralisation (MIH).' The GRADE system was used to assess the quality of evidence regarding aetiology and treatment which was judged as HIGH, MODERATE, LOW or VERY LOW, while the GRADE criteria were used to indicate the strength of recommendation regarding treatment options as STRONG or WEAK/CONDITIONAL. Results: (A) Regarding aetiology, it is confirmed that MIH has a multifactorial aetiology with the duration, strength and timing of occurrence of the aetiological factors being responsible for the variable clinical characteristics of the defect. Perinatal hypoxia, prematurity and other hypoxia related perinatal problems, including caesarean section, appear to increase the risk of having MIH, while certain infant and childhood illnesses are also linked with MIH. In addition, genetic predisposition and the role of epigenetic influences are becoming clearer following twin studies and genome and single-nucleotide polymorphisms analyses in patients and families. Missing genetic information might be the final key to truly understand MIH aetiology. (B) Regarding treatment options, composite restorations, preformed metal crowns and laboratory indirect restorations provide high success rates for the posterior teeth in appropriate cases, while scheduled extractions provide an established alternative option in severe cases. There is great need for further clinical and laboratory studies evaluating new materials and non-invasive/micro-invasive techniques for anterior teeth, especially when aesthetic and oral health related quality of life (OHRQoL) issues are concerned. Conclusions: MIH has been studied more extensively in the last decade. Its aetiology follows the multifactorial model, involving systemic medical and genetic factors. Further focused laboratory research and prospective clinical studies are needed to elucidate any additional factors and refine the model. Successful preventive and treatment options have been studied and established. The appropriate choice depends on the severity of the defects and the age of the patient. EAPD encourages the use of all available treatment options, whilst in severe cases, scheduled extractions should be considered. [ABSTRACT FROM AUTHOR]

Purpose: To systematically review the treatment modalities for molar-incisor hypomineralisation for children under the age of 18 years. The research question was, 'What are the treatment options for teeth in children affected by molar incisor hypomineralisation?' Methods: An electronic search of the following electronic databases was completed MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, LILACS, Google Scholar and Open Grey identifying studies from 1980 to 2020. The PRISMA guidelines were followed. The studies were screened, data extracted and calibration was completed by two independent reviewers. Results: Of 6220 potential articles, 34 studies were included. Twenty studies investigated management of molars with fissure sealants, glass ionomer cement, polyacid modified resin composite, composite resin, amalgam, preformed metal crowns, laboratory-manufactured crowns and extractions. In four articles management of incisors with microabrasion, resin-infiltration and a combination of approaches was reported. Eight studies looked at strategies to mineralise MIH-affected teeth and/or reduce hypersensitivity. Two studies investigated patient-centred outcomes following treatment. Due to the heterogeneity between the studies, meta-analysis was not performed. Conclusion: The use of resin-based fissure sealants, preformed metal crowns, direct composite resin restorations and laboratory-made restorations can be recommended for MIH-affected molars. There is insufficient evidence to support specific approaches for the management of affected incisors. Products containing CPP-ACP may be beneficial for MIH-affected teeth. [ABSTRACT FROM AUTHOR]

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5′ UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5′ UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5′ UTRs. A total of ~12,000 5′ UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5′ UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5′ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies. The engineering of 5′ UTRs that modulate protein expression remains a great challenge. Here we leverage synthetic biology and computational design to develop a high-throughput strategy to design, screen, and optimize 5′ UTRs that enhance protein expression for non-viral gene therapies. [ABSTRACT FROM AUTHOR]

The systematic functional analysis of combinatorial genetics has been limited by the throughput that can be achieved and the order of complexity that can be studied. To enable massively parallel characterization of genetic combinations in human cells, we developed a technology for rapid, scalable assembly of high-order barcoded combinatorial genetic libraries that can be quantified with high-throughput sequencing. We applied this technology, combinatorial genetics en masse (CombiGEM), to create high-coverage libraries of 1,521 two-wise and 51,770 three-wise barcoded combinations of 39 human microRNA (miRNA) precursors. We identified miRNA combinations that synergistically sensitize drug-resistant cancer cells to chemotherapy and/or inhibit cancer cell proliferation, providing insights into complex miRNA networks. More broadly, our method will enable high-throughput profiling of multifactorial genetic combinations that regulate phenotypes of relevance to biomedicine, biotechnology and basic science. [ABSTRACT FROM AUTHOR]

The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB participate in diverse neuronal functions, including activity-dependent synaptic plasticity that is crucial for learning and memory. On binding to BDNF, TrkB is not only autophosphorylated at tyrosine residues but also undergoes serine phosphorylation at S478 by the serine/threonine kinase cyclin-dependent kinase 5 (Cdk5). However, the in vivo function of this serine phosphorylation remains unknown. We generated knock-in mice lacking this serine phosphorylation (TrkbS478A/S478A mice) and found that the TrkB phosphorylation-deficient mice displayed impaired spatial memory and compromised hippocampal long-term potentiation (LTP). S478 phosphorylation of TrkB regulates its interaction with the Rac1-specific guanine nucleotide exchange factor TIAM1, leading to activation of Rac1 and phosphorylation of S6 ribosomal protein during activity-dependent dendritic spine remodeling. These findings reveal the importance of Cdk5-mediated S478 phosphorylation of TrkB in activity-dependent structural plasticity, which is crucial for LTP and spatial memory formation. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that is increasingly implicated in various neurodegenerative diseases. Deregulated Cdk5 activity has been associated with neuronal death, but the underlying mechanisms are not well understood. Here we report an unexpected role for Cdk5 in the regulation of induced autophagy in neurons. We have identified endophilin B1 (EndoB1) as a Cdk5 substrate, and show that Cdk5-mediated phosphorylation of EndoB1 is required for autophagy induction in starved neurons. Furthermore, phosphorylation of EndoB1 facilitates EndoB1 dimerization and recruitment of UVRAG (UV radiation resistance-associated gene). More importantly, Cdk5-mediated phosphorylation of EndoB1 is essential for autophagy induction and neuronal loss in models of Parkinson's disease. Our findings not only establish Cdk5 as a critical regulator of autophagy induction, but also reveal a role for Cdk5 and EndoB1 in the pathophysiology of Parkinson's disease through modulating autophagy. [ABSTRACT FROM AUTHOR]