A5002404839- CRISPR and Genetic Engineering
- RNA and protein synthesis mechanisms
- Advanced biosensing and bioanalysis techniques
- Insect Resistance and Genetics
- Immunotherapy and Immune Responses
- Signaling Pathways in Disease
- Genetics, Aging, and Longevity in Model Organisms
- vaccines and immunoinformatics approaches
- Pluripotent Stem Cells Research
- Monoclonal and Polyclonal Antibodies Research
- RNA Interference and Gene Delivery
- Antimicrobial Resistance in Staphylococcus
- Innovation and Socioeconomic Development
- Transgenic Plants and Applications
- Bacterial Genetics and Biotechnology
- Antimicrobial Peptides and Activities
- Innovative Microfluidic and Catalytic Techniques Innovation
- Mosquito-borne diseases and control
- Insect symbiosis and bacterial influences
- Bacterial biofilms and quorum sensing
National University of Singapore
2024
ToolGen (South Korea)
2017-2023
Stanford University
2008
Howard Hughes Medical Institute
2008
University of Chicago
2008
University of Cambridge
2004
Abstract The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated often poorly efficient in human cells. Here, we develop directed evolution approach E. coli to obtain Sniper-Cas9, which shows high without killing on-target activities Unlike other engineered variants, Sniper-Cas9 WT-level with...
Abstract Although several high-fidelity SpCas9 variants have been reported, it has observed that this increased specificity is associated with reduced on-target activity, limiting the applications of when efficient genome editing required. Here, we developed an improved version Sniper–Cas9, Sniper2L, which represents exception to trade-off trend as showed higher retained high activity. We evaluated Sniper2L activities at a large number target sequences and DeepSniper, deep learning model can...
Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be challenge. Non-canonical gRNAs and new engineered variants have been developed improve specificity, but at the cost on-target activity. DNA unwinding is checkpoint before cleavage by Cas9, was shown more sensitive sequence mismatches specificity-enhancing mutations in Cas9s. Here we performed single-molecule FRET-based experiments using various combinations non-canonical different All Cas9s...
The human pathogen Pseudomonas aeruginosa uses N-butyryl-L-homoserine lactone (BHL) and N-(3-oxododecanyl)-L-homoserine (OdDHL) as small molecule intercellular signals in a phenomenon known quorum sensing (QS). QS modulators are effective at attenuating P. virulence; therefore, they potential new class of antibacterial agent. BHL OdDHL is hydrolysed under physiological conditions. hydrolysis proceeds rate faster than racemisation the α-chiral centre. Non-hydrolysable, non-racemic analogues...
Abstract Prime editors (PEs) are powerful tools that widen the possibilities for sequence modifications during genome editing. Although methods based on analysis of Cas9 nuclease or nickase activity have been used to predict genome-wide off-target activities PEs, no tool directly uses PEs this purpose has reported yet. In study, we present a cell-based assay, named TAgmentation Editor sequencing (TAPE-seq), provides candidates PEs. TAPE-seq analyses successfully performed using many...
We present a novel genome-wide off-target prediction method named Extru-seq and compare it with cell-based (GUIDE-seq), in vitro (Digenome-seq), silico methods using promiscuous guide RNAs large numbers of valid sites. demonstrates high validation rate retention information about the intracellular environment, both beneficial characteristics methods. also shows low miss could easily be performed clinically relevant cell types little optimization, which are major positive features In summary,
Clustered regularly interspaced short palindromic repeats (CRISPR)-based editing tools have transformed the landscape of genome editing. However, absence a robust and safe CRISPR delivery method continues to limit its potential for therapeutic applications. Despite emergence various methodologies aimed at addressing this challenge, issues regarding efficiency operations persist. We introduce microfluidic gene system, called droplet cell pincher (DCP), designed highly efficient This approach...
Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how mediate catalysis requires elucidation of ion interactions with both the enzyme and substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish interrogate transition interactions. Tetrahymena group I ribozyme is paradigm study RNA...
The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance its potentially deleterious off-target effects. Several methods have been devised to reduce such Here, we present an Escherichia coli-based directed evolution method called Sniper-screen obtain a Cas9 variant with optimized specificity and retained on-target activity, Sniper-Cas9. Using Sniper-screen, positive negative selection...
While computational epitope prediction methods have found broad application, their use, specifically in allergy-related contexts, remains relatively less explored. This study benchmarks several publicly available tools, focusing on the allergenic IgE and T-cell epitopes of Fel d 1, an extensively studied allergen. Using a variety tools accessible via Immune Epitope Database (IEDB) other resources, we evaluate ability to identify known linear 1. Our results show limited effectiveness for...
Abstract The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated often poorly efficient in human cells. Here, we have used directed evolution approach E. coli to obtain Sniper-Cas9, which shows high without sacrificing on-target activities
ABSTRACT Cas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be challenge. Non-canonical gRNAs and new engineered variants have been developed improve but at the cost on-target activity. DNA unwinding is primary checkpoint before cleavage by was shown more sensitive sequence mismatches specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based experiments using various combinations non-canonical different...
The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance its potentially deleterious off-target effects. Several methods have been devised to reduce such Here, we present an Escherichia coli-based directed evolution method called Sniper-screen obtain a Cas9 variant with optimized specificity and retained on-target activity, Sniper-Cas9. Using Sniper-screen, positive negative selection...
Abstract While computational epitope prediction methods have found broad application, their use, specifically in allergy-related contexts, remains relatively less explored. This study benchmarks several publicly available tools, focusing on the allergenic IgE and T-cell epitopes of Fel d 1, an extensively studied allergen. Using a variety tools accessible via Immune Epitope Database (IEDB) other resources, we evaluate ability to identify known linear 1. Our results show limited effectiveness...
Abstract Although several high-fidelity SpCas9 variants that have reduced activities at mismatched target sequences been reported, it has observed this increased specificity is associated with on-target activity, limiting the applications of when efficient genome editing required. Here, we developed an improved version Sniper-Cas9, Sniper2L, which represents exception to trade-off trend as showed higher retained high activity. We evaluated Sniper2L a large number sequences, and DeepSniper,...