Project description:Cellular RNA is decorated with over 160 types of chemical modifications. Many modifications in mRNA, including m6A and m5C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that could modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq).
Project description:Cellular RNA is decorated with over 160 types of chemical modifications. Many modifications in mRNA, including m6A and m5C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that could modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq).
Project description:Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.
Project description:Diterpenoids and sucrose esters are synthesized in cigar tobacco leave (CTLs) trichomes and secreted onto leaf surface as trichome exudate (TE), contributing to sensory quality.To reveal the mechanism by which TE affects CTLs quality, this study employed the half-leaf method, utilizing tobacco extract as the fermentation medium for cigar tobacco leaves. We analyze the effects of TE on the sensory quality, chemical composition, and microbial communities of cigar tobacco leaves. The levels of degradation products (solanone, isovaleric acid, sclareolide, and sclareol) in the trichome exudate increased, while the levels of reducing sugars, proteins, and tobacco alkaloids decreased. These chemical changes are key factors in enhancing the aroma of smoke, increasing sweetness, reducing harshness, and lowering physiological strength. Metabolic alterations in tobacco leaves led to significantly increased levels of lipids and lipoids, organic acids and their derivatives, and organic heterocyclic compounds. TE enriches Pseudomonas aff. cichorii and Aspergillus aff. intermedius, which degrade nicotine and protein, increase the contents of reducing sugars, amino acids, lipids and lipid-like molucules. This reduces the irritation and physiological strength of CTLs and enhances the sweet taste. Meanwhile, TE is degraded and transformed by P. aff. cichorii to generate aroma compounds, thereby enhancing the aroma quality of cigar tobacco leaves.This study demonstrated that TE not only provides aroma precursors but also modifies leaf chemical components by shaping the microbial community during fermentation, thereby improving CTLs quality and providing a theoretical basis for establishing an artificial fermentation technology system for CTLs based on TE.