EcCas6e-based antisense crRNA for gene repression and RNA editing in microorganisms.

Precise gene regulation and programmable RNA editing are vital RNA-level regulatory mechanisms. Gene repression tools grounded in small non-coding RNAs, microRNAs, and CRISPR-dCas proteins, along with RNA editing tools anchored in Adenosine Deaminases acting on RNA (ADARs), have found extensive application in molecular biology and cellular engineering. Here, we introduced a novel approach wherein we developed an EcCas6e mediated crRNA-mRNA annealing system for gene repression in Escherichia coli and RNA editing in Saccharomyces cerevisiae.

PAMless SpRY exhibits a preference for the seed region for efficient targeting.

Protospacer-adjacent motif (PAM) recognition licenses Cas nucleases for genome engineering applications, thereby restricting gene targeting to PAM-containing regions. Protein engineering has led to PAM-relaxed SpCas9 variants like SpG and SpRY. Given the evolved role of PAMs in facilitating target-searching kinetics, it remains unclear how these variants quickly locate their targets.

Recent advances in CRISPR-Cas9-based genome insertion technologies.

Programmable genome insertion (or knock-in) is vital for both fundamental and translational research. The continuously expanding number of CRISPR-based genome insertion strategies demonstrates the ongoing development in this field. Common methods for site-specific genome insertion rely on cellular double-strand breaks repair pathways, such as homology-directed repair, non-homologous end-joining, and microhomology-mediated end joining.

Integrating thermodynamic and enzymatic constraints into genome-scale metabolic models.

Stoichiometric genome-scale metabolic network models (GEMs) have been widely used to predict metabolic phenotypes. In addition to stoichiometric ratios, other constraints such as enzyme availability and thermodynamic feasibility can also limit the phenotype solution space. Extended GEM models considering either enzymatic or thermodynamic constraints have been shown to improve prediction accuracy.

DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing.

Many clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based genome editing technologies take advantage of Cas nucleases to induce DNA double-strand breaks (DSBs) at desired locations within a genome. Further processing of the DSBs by the cellular DSB repair machinery is then necessary to introduce desired mutations, sequence insertions, or gene deletions. Thus, the accuracy and efficiency of genome editing are influenced by the cellular DSB repair pathways.

Clutch mechanism of chemomechanical coupling in a DNA resecting motor nuclease.

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3' to 5' translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet-like fashion.

RADX controls RAD51 filament dynamics to regulate replication fork stability.

The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication.

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates.

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA).

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood.

Human Condensin I and II Drive Extensive ATP-Dependent Compaction of Nucleosome-Bound DNA.

Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA.

Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection.

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease.

Single-molecule visualization of human BLM helicase as it acts upon double- and single-stranded DNA substrates.

Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR).

The RecQ helicase Sgs1 drives ATP-dependent disruption of Rad51 filaments.

DNA helicases of the RecQ family are conserved among the three domains of life and play essential roles in genome maintenance. Mutations in several human RecQ helicases lead to diseases that are marked by cancer predisposition. The Saccharomyces cerevisiae RecQ helicase Sgs1 is orthologous to human BLM, defects in which cause the cancer-prone Bloom's Syndrome.

Mechanisms of Type I-E and I-F CRISPR-Cas Systems in .

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invasion by bacteriophages and other mobile genetic elements. Short fragments of invader DNA are stored as immunological memories within CRISPR (clustered regularly interspaced short palindromic repeat) arrays in the host chromosome. These arrays provide a template for RNA molecules that can guide CRISPR-associated (Cas) proteins to specifically neutralize viruses upon subsequent infection.

Fluorescence-based methods for measuring target interference by CRISPR-Cas systems.

Type I, II, and V CRISPR-Cas systems are RNA-guided dsDNA targeting defense mechanisms found in bacteria and archaea. During CRISPR interference, Cas effectors use CRISPR-derived RNAs (crRNAs) as guides to bind complementary sequences in foreign dsDNA, leading to the cleavage and destruction of the DNA target. Mutations within the target or in the protospacer adjacent motif can reduce the level of CRISPR interference, although the level of defect is dependent on the type and position of the mutation, as well as the guide sequence of the crRNA.

Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade.

CRISPR-Cas systems defend bacteria and archaea against infection by bacteriophage and other threats. The central component of these systems are surveillance complexes that use guide RNAs to bind specific regions of foreign nucleic acids, marking them for destruction. Surveillance complexes must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear.

Conformational Control of Cascade Interference and Priming Activities in CRISPR Immunity.

During type I-E CRISPR-Cas immunity, the Cascade surveillance complex utilizes CRISPR-derived RNAs to target complementary invasive DNA for destruction. When invader mutation blocks this interference activity, Cascade instead triggers rapid primed adaptation against the invader. The molecular basis for this dual Cascade activity is poorly understood.

CRISPR interference and priming varies with individual spacer sequences.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring 'spacer' sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid 'primed' adaptation.

Suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in Saccharopolyspora spinosa.

Background: Polyketides, such as spinosad, are mainly synthesized in the stationary phase of the fermentation. The synthesis of these compounds requires many primary metabolites, such as acetyl-CoA, propinyl-CoA, NADPH, and succinyl-CoA. Their synthesis is also significantly influenced by NADH/NAD+.