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Panoptes, an anti-phage defence system against virus-mediated immune suppression, is revealed.

How do large RNA molecules find their active conformations among a universe of possible structures? Two recent studies reveal that RNA folding is a rapid and ordered process, with surprising similarities to protein folding mechanisms.

Nature Biotechnology asks selected members of the international community to comment on the ethical issues raised by the prospect of CRISPR-Cas9 engineering of the human germline.

Genome-editing presents many opportunities. But the advent of human-germline editing brings urgency to ethical discussions, says Jennifer Doudna.

Nature Biotechnology asks a selection of researchers about the most exciting frontier in their field and the most needed technologies for advancing knowledge and applications.

The bacterial CRISPR/Cas system acquires short phage sequences known as spacers that integrate between CRISPR repeats and constitute a record of phage infection; this study shows that the Cas1–Cas2 complex is the minimal machinery required for spacer acquisition and the complex integrates oligonucleotide DNA substrates into acceptor DNA in a manner similar to retroviral integrases and DNA transposases with Cas 1 as the catalytic subunit and Cas2 acting to increase integration activity.

Jennifer Doudna, a pioneer of the revolutionary genome-editing technology, reflects on how 2015 became the most intense year of her career — and what she's learnt.

Structural comparison of predicted viral protein structures with known protein structures suggests taxonomic relationships and functions for up to 25% of unannotated viral proteins, including many with putative functions in host immune evasion.

The structure of the Cas1–Cas2 complex bound to a protospacer sequence illustrates how foreign DNA is captured and measured by bacterial proteins in preparation for integration into CRISPR loci.

Cell-specific molecular delivery with enveloped delivery vehicles enables genome editing ex vivo and in vivo.

The delivery of CRISPR RNPs has potential advantages over other genome editing approaches, including reduced off-target editing and reduced immunogenicity. Here the authors report self-deliverable Cas9 RNPs capable of robustly editing cultured cells in vitro and the mouse brain upon direct injections.

A fluorescence-based approach defines new features of Cas9 that control the specificity of RNA-guided DNA cleavage in CRISPR genome editing technology.

The CRISPR-associated bacterial enzyme C2c2 is shown to contain two separable, distinct sites for the highly sensitive detection and cleavage of single-stranded RNA.

In the presence of a short DNA oligonucleotide containing a protospacer adjacent motif, a guide-RNA-programmed Cas9 is able to specifically bind and/or cleave single-stranded RNA—this system can be used to isolate specific endogenous RNA transcripts from a cell lysate without any tag or modification.

Using a metagenomic approach, three types of CRISPR–Cas systems have been discovered in uncultivated bacterial and archaeal hosts from a variety of different environments.

A new engineered version of SpCas9, called HypaCas9, displays enhanced accuracy of editing without significant loss of efficiency at the desired target.

Doudna and colleagues determine the mechanisms used by type V anti-CRISPR proteins. AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the Cas12a-bound guide RNA, while AcrVA4 and AcrVA5 inhibit recognition of dsDNA.

Leveraging RNA-targeting dCas13d enables selective interference with phage protein translation and facilitates measurement of phage gene fitness at a transcriptome-wide scale.

The CRISPR–Cas system mediates immunity to foreign DNA sequences that are integrated as spacers between repeats in the CRISPR locus. Work from Doudna and colleagues shows that nucleases Cas1 and Cas2 form a stable complex that recognizes the CRISPR leader-repeat sequence, thus determining the site of integration.

This study presents a protein search framework with conformal prediction, enabling statistically reliable annotation of protein function. The method improves homology search, enzyme classification, and filters proteins for further characterization.

Generating RNA sequences with improved function remains challenging. Here, authors present an RNA database for RNA structural and functional analysis. They use this database and the RNA generative models to identify RNA mutations that increase the thermostability of a bacterial ribosome.

Hepatitis C virus (HCV) uses an internal ribosome entry site (IRES) to directly recruit ribosomes to the viral mRNA translation-start site. Recent structural studies and biochemical data reveal how the HCV IRES interacts with the 40S ribosomal subunit and the cellular protein-synthesis machinery to direct efficient translation initiation.

An engineered clustered regularly interspaced short palindromic repeat ribonucleoprotein delivered in lipid nanoparticles efficiently edits cells in vivo.

Single-molecule live-cell fluorescence in situ hybridization uses an RNA-targeting CRISPR–Csm complex to image and track endogenous RNAs.

Recent technological advances have enabled the probing of RNA structure across the transcriptomes of various speciesin vitro and in vivo. This Review discusses our latest understanding of how RNA structure influences various steps of gene expression, including translation, mRNA localization and microRNA-mediated gene regulation.

CRISPR systems lacking Cas4 can use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.

A COVID-19 test implemented in an automated microfluidic device and leveraging isothermal RNA amplification followed by T7 transcription and Cas13-mediated cleavage of a quenched fluorophore rapidly detects SARS-CoV-2 RNA in saliva samples.

A CRISPR-Cas13 enzyme is shown to have potent anti-phage activity that is harnessed to produce a phage genome engineering method with broad utility.

The bacterial Csm complex efficiently knocks down eukaryotic nuclear and cytoplasmic RNAs.

Crystal structures of unprocessed and mature crRNA-bound LbaCas13a shed light upon catalytic residues involved in crRNA maturation and mechanisms blocking Cas13a nuclease activity before target-RNA binding.

A suite of methods enables programmable species- and locus-specific editing of bacteria in communities.

CRISPR-Cas12a is a programmable endonuclease used for genetic engineering. Here, the authors use real-time single-molecule measurements to show that Cas12a unwinds the target DNA site in dynamic and reversible steps to test for matches with its guide RNA molecule.

In some systems, a single protein comprising reverse transcriptase (RT), integrase and maturase enables concerted sequence integration and crRNA production. Here, analyses including the structure of a Cas6-RT-Cas1—Cas2 complex suggest coordination between all three active sites and capacity to acquire CRISPR sequences from RNA and DNA substrates.

Omicron BA.1 is attenuated in infection models though the precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging. Here the authors present pGLUE, a plasmid-based viral genome assembly and rescue strategy, to systematically characterize Omicron mutations and show that Omicron NSP6 has weakened lipid flow to replication organelles and reduced viral RNA replication.

Type III CRISPR-Cas systems are able to target transcriptionally active DNA sequences in phages and plasmids. Here, the authors reveal the mechanism of the target nucleic acid preference of Type III-A CRISPR-Cas complexes at the transcription bubble by a combination of structural and biochemical approaches.

CRISPR-Cas9 has enabled an unprecedented ability to manipulate the genome yet it is still poorly understood how target recognition functions at a molecular level. Here the authors use single-molecule FRET to probe Cas9-target interaction and identify distinct search and proofreading states.

A bacterial enzyme that uses guide RNA molecules to target DNA for cleavage has been adopted as a programmable tool to site-specifically modify genomes of cells and organisms, from bacteria and human cells to whole zebrafish.

Cryo-EM structures of CRISPR–CasΦ, a small RNA-guided enzyme unique to bacteriophages, reveal how CasΦ binds to and cleaves DNA, paving the way for engineering of improved CasΦ variants for diagnostics and genome-editing applications.

This Perspective discusses how the Somatic Cell Genome Editing Consortium aims to accelerate the implementation of safe and effective genome-editing therapies in the clinic.