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CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.
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http://dx.doi.org/10.1186/s13059-023-03132-3 | DOI Listing |
J Agric Food Chem
September 2025
State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Provincial Key Laboratory of Microbial Molecular Biology, College of Life Science, Hunan Normal University, Changsha 410081, China.
Entomopathogenic nematode symbiotic bacteria (EPNB) enhance nematode insecticidal capacity through symbiosis. This study cloned the complete 32-kb type III secretion system (T3SS) gene cluster from TT01 using Red/ET recombineering and functionally expressed it in T3SS-deficient HN_xs01. Heterologous T3SS expression significantly enhanced HN_xs01 adhesion and invasion capabilities in CF-203 cells.
View Article and Find Full Text PDFmSystems
September 2025
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
A significant challenge in the field of microbiology is the functional annotation of novel genes from microbiomes. The increasing pace of sequencing technology development has made solving this challenge in a high-throughput manner even more important. Functional metagenomics offers a sequence-naive and cultivation-independent solution.
View Article and Find Full Text PDFIn this work, we present a streamlined one-pot cloning and protein expression platform that integrates mutagenesis, plasmid assembly, and functional protein testing in a single reaction. By combining Golden Gate cloning with cell-free transcription-translation, we demonstrate efficient generation and screening of genetic variants without the need for intermediate purification or bacterial amplification. Using fluorescent proteins, luciferase enzymes, antibiotic-converting enzymes, and the violacein biosynthetic pathway, we validate the versatility of this approach for single-and multi-site mutagenesis, combinatorial variant libraries, metabolic pathway programming, and whole-plasmid assembly.
View Article and Find Full Text PDFFront Immunol
September 2025
Kling Biotherapeutics, Amsterdam, Netherlands.
Introduction: Amid the persistent threat of future pandemics, the continuous evolution of SARS-CoV-2 exposed critical challenges for vaccine efficacy and therapeutic interventions, highlighting the need for rapid and adaptable approaches to respond to immune escape variants.
Methods: Here, we report the use of immortalized B cell libraries from human peripheral blood mononuclear cells (PBMCs) and tonsil tissues to uncover B cell clones exhibiting cross-reactive neutralization against various SARS-CoV-2 variants and perform directed evolution of immortalized B cell clones to produce antibodies with improved binding and neutralization against emerging SARS-CoV-2 variants.
Results: Immortalization of PBMC and tonsil-derived human B cells was achieved through transduction with retroviral vectors encoding apoptosis inhibitors, yielding transduction efficiencies of 67.
Bioinformatics
September 2025
Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, 2333 ZA, the Netherlands.
Motivation: The generation and analysis of diverse mutants of a protein is a powerful tool for understanding protein function. However, generating such mutants can be time-consuming, while the commercial option of buying a series of mutant plasmids can be expensive. In contrast, the insertion of a synthesized double stranded DNA (dsDNA) fragment into a plasmid is a fast and low-cost method to generate a large library of mutants with one or more point mutations, insertions, or deletions.
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