Type III-B CRISPR-Cas cascade of proteolytic cleavages.

The generation of cyclic oligoadenylates and subsequent allosteric activation of proteins that carry sensory domains is a distinctive feature of type III CRISPR-Cas systems. In this work, we characterize a set of associated genes of a type III-B system from that contains two caspase-like proteases, SAVED-CHAT and PCaspase (prokaryotic caspase), co-opted from a cyclic oligonucleotide-based antiphage signaling system (CBASS). Cyclic tri-adenosine monophosphate (AMP)-induced oligomerization of SAVED-CHAT activates proteolytic activity of the CHAT domains, which specifically cleave and activate PCaspase.

Characterization of the AcrIIC1 anti‒CRISPR protein for Cas9‒based genome engineering in E. coli.

Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli.

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a.

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools.

Efficient Genome and Base Editing in Human Cells Using ThermoCas9.

Most genetic engineering applications reported thus far rely on the type II-A CRISPR-Cas9 nuclease from (SpyCas9), limiting the genome-targeting scope. In this study, we demonstrate that a small, naturally accurate, and thermostable type II-C Cas9 ortholog from (ThermoCas9) with alternative target site preference is active in human cells, and it can be used as an efficient genome editing tool, especially for gene disruption. In addition, we develop a ThermoCas9-mediated base editor, called ThermoBE4, for programmable nicking and subsequent C-to-T conversions in human genomes.

Cryo-EM structure of the transposon-associated TnpB enzyme.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide.

Revealing determinants of translation efficiency via whole-gene codon randomization and machine learning.

It has been known for decades that codon usage contributes to translation efficiency and hence to protein production levels. However, its role in protein synthesis is still only partly understood. This lack of understanding hampers the design of synthetic genes for efficient protein production.

The genome editing revolution.

A series of spectacular scientific discoveries and technological advances in the second half of the 20th century have provided the basis for the ongoing genome editing revolution. The elucidation of structural and functional features of DNA and RNA was followed by pioneering studies on genome editing: Molecular biotechnology was born. Since then, four decades followed during which progress of scientific insights and technological methods continued at an overwhelming pace.

Compact but mighty: Biology and applications of type III-E CRISPR-Cas systems.

In this issue, Liu et al. present an in-depth study aiming to unravel the structural, biochemical, and physiological aspects of how type III-E CRISPR-Cas systems trigger abortive infection by activating a protease upon target RNA recognition..

The miniature CRISPR-Cas12m effector binds DNA to block transcription.

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M.

Metabolic engineering of Clostridium autoethanogenum for ethyl acetate production from CO.

Background: Ethyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO/H, is desirable.

The double life of CRISPR-Cas13.

Since the discovery of RNA-programmable nucleases from the prokaryotic adaptive immune system CRISPR-Cas, these proteins have seen rapid and widespread adoption for biotechnological and clinical research. A recently discovered system, CRISPR-Cas13, uses CRISPR RNA guides to target RNA. Interestingly, RNA targeting by Cas13 results in cleavage of both target RNA and bystander RNA.

Comparative Metagenomic Analysis of Biosynthetic Diversity across Sponge Microbiomes Highlights Metabolic Novelty, Conservation, and Diversification.

Marine sponges and their microbial symbiotic communities are rich sources of diverse natural products (NPs) that often display biological activity, yet little is known about the global distribution of NPs and the symbionts that produce them. Since the majority of sponge symbionts remain uncultured, it is a challenge to characterize their NP biosynthetic pathways, assess their prevalence within the holobiont, and measure the diversity of NP biosynthetic gene clusters (BGCs) across sponge taxa and environments. Here, we explore the microbial biosynthetic landscapes of three high-microbial-abundance (HMA) sponges from the Atlantic Ocean and the Mediterranean Sea.

Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides.

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity.

Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates Conserved Protospacer Selection Mechanisms Between Diverse CRISPR-Cas Types.

Adaptation of clustered regularly interspaced short palindromic repeats (CRISPR) arrays is a crucial process responsible for the unique, adaptive nature of CRISPR-Cas immune systems. The acquisition of new CRISPR spacers from mobile genetic elements has previously been studied for several types of CRISPR-Cas systems. In this study, we used a high-throughput sequencing approach to characterize CRISPR adaptation of the type V-A system from and the type V-B system from .

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations.

Alternative functions of CRISPR-Cas systems in the evolutionary arms race.

CRISPR-Cas systems of bacteria and archaea comprise chromosomal loci with typical repetitive clusters and associated genes encoding a range of Cas proteins. Adaptation of CRISPR arrays occurs when virus-derived and plasmid-derived sequences are integrated as new CRISPR spacers. Cas proteins use CRISPR-derived RNA guides to specifically recognize and cleave nucleic acids of invading mobile genetic elements.

A Hyperthermoactive-Cas9 Editing Tool Reveals the Role of a Unique Arsenite Methyltransferase in the Arsenic Resistance System of Thermus thermophilus HB27.

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to humans. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (SmtB). Here, we characterize the arsenic resistance system of T.

Comprehensive Genome Engineering Toolbox for Microalgae Based on CRISPR-Cas Systems.

Microalgae can produce industrially relevant metabolites using atmospheric CO and sunlight as carbon and energy sources, respectively. Developing molecular tools for high-throughput genome engineering could accelerate the generation of tailored strains with improved traits. To this end, we developed a genome editing strategy based on Cas12a ribonucleoproteins (RNPs) and homology-directed repair (HDR) to generate scarless and markerless mutants of the microalga .

Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas.

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas).

SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation.

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA.

Towards a synthetic cell cycle.

Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives.