Rationally designed pooled CRISPRi-seq uncovers an inhibitor of bacterial peptidyl-tRNA hydrolase.

Bacterial mutant libraries with downregulated antibiotic targets are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi).

Rationally Designed Pooled CRISPRi-Seq Uncovers an Inhibitor of Bacterial Peptidyl-tRNA Hydrolase.

Unlabelled: Pooled knockdown libraries of essential genes are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we describe the construction of CIMPLE ( C RISPR i - m ediated p ooled library of e ssential genes), a rationally designed pooled knockdown library built in a model antibiotic-resistant bacteria, By analyzing growth parameters of clonal knockdown populations of an arrayed CRISPRi library, we predicted strain depletion levels during pooled growth and adjusted mutant relative abundance, approaching genomic coverage of antibacterial targets during antibiotic exposure.

Optically Controlled CRISPR-Cas9 and Cre Recombinase for Spatiotemporal Gene Editing: A Review.

CRISPR-Cas9 and Cre recombinase, two tools extensively used for genome interrogation, have catalyzed key breakthroughs in our understanding of complex biological processes and diseases. However, the immense complexity of biological systems and off-target effects hinder clinical applications, necessitating the development of platforms to control gene editing over spatial and temporal dimensions. Among the strategies developed for inducible control, light is particularly attractive as it is noninvasive and affords high spatiotemporal resolution.

Identification of sequence determinants for the ABHD14 enzymes.

Over the course of evolution, enzymes have developed remarkable functional diversity in catalyzing important chemical reactions across various organisms, and understanding how new enzyme functions might have evolved remains an important question in modern enzymology. To systematically annotate functions, based on their protein sequences and available biochemical studies, enzymes with similar catalytic mechanisms have been clustered together into an enzyme superfamily. Typically, enzymes within a superfamily have similar overall three-dimensional structures, conserved catalytic residues, but large variations in substrate recognition sites and residues to accommodate the diverse biochemical reactions that are catalyzed within the superfamily.