One-pot universal NicE-seq: all enzymatic downstream processing of 4% formaldehyde crosslinked cells for chromatin accessibility genomics

Background Accessible chromatin landscape allows binding of transcription factors, and remodeling of promoter and enhancer elements during development. Chromatin accessibility along with integrated multiomics approaches have been used for determining molecular subtypes of cancer in patient samples. Results One-pot Universal NicE-seq (One-pot UniNicE-seq) is an improved accessible chromatin profiling method that negate DNA purification and incorporate sonication free enzymatic fragmentation before library preparation and is suited to a variety of mammalian cells. One-pot UniNicE-seq is versatile, capable of profiling 4% formaldehyde fixed chromatin in as low as 25 fixed cells. Accessible chromatin profile is more efficient on formaldehyde-fixed cells using one-pot UniNicE-seq compared to Tn5 transposon mediated methods, demonstrating its versatility. Conclusion One-pot UniNicE-seq allows the entire process of accessible chromatin labeling and enrichment in one pot at 4% formaldehyde cross-linking conditions. It doesn’t require enzyme titration, compared to other technologies, since accessible chromatin is labelled with 5mC incorporation and deter degradation by nicking enzyme, thus opening the possibility for automation.

Directed Evolution of Enzymes based on in vitro Programmable Self-Replication

High-throughput directed evolution, implemented in well-controlled in vitro conditions, provides a powerful route for enzyme engineering. Most existing technologies are based on activity screening and require the sequential observation and sorting of each individual variant. By contrast, approaches based on autonomous feedback loops, linking phenotype to genotype replication, enable autonomous selection without screening. However, these approaches are only possible in vivo, or applicable to very specific activities, such as polymerases or ligases. Here, we leverage synthetic molecular networks to create a programmable in vitro feedback loop linking a target enzymatic activity to gene amplification. After encapsulation and lysis of up to 10^7 transformed variants, the genes present in each droplet are amplified according to the activity of the encoded enzyme, resulting in the autonomous enrichment of interesting sequences. Applied to a nicking enzyme with thermal or kinetic selection pressures, this method reveals detailed mutational landscapes and provides improved variants.

A highly sensitive fluorescence biosensor for detection of Staphylococcus aureus based on HCR-mediated three-way DNA junction nicking enzyme assisted signal amplification

Sensitive and efficient monitoring of food-borne bacteria is of great importance for food safety control. Herein, a novel biosensor for highly sensitive detection of Staphylococcus aureus (S. aureus) was constructed...

A nucleotide-sensing endonuclease from the Gabija bacterial defense system

ABSTRACTThe arms race between bacteria and phages has led to the development of exquisite bacterial defense systems including a number of uncharacterized systems distinct from the well-known Restriction-Modification and CRISPR/Cas systems. Here, we report functional analyses of the GajA protein from the newly predicted Gabija system. The GajA protein is revealed as an endonuclease unique in that: 1. It may function as a restriction enzyme or a site-specific nicking enzyme, depending on the arrangement of the recognition sequences; 2. Its activity is strictly regulated by nucleotides concentration. NTP and dNTP at physiological concentrations can fully inhibited the robust DNA cleavage activity of GajA. Interestingly, the nucleotide inhibition is mediated by an ATPase-like domain, which usually hydrolyzes ATP to stimulate the DNA cleavage when associated with other nucleases. These features suggested the mechanism of the Gabija defense in which an endonuclease activity was suppressed at normal condition, while activated by the depletion of NTP and dNTP upon the replication and transcription by invaded phages. This work highlights a concise strategy to utilize a single protein for phage resistance via nucleotide regulatory.