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Cryo-electron microscopy structures of Streptococcus pyogenes Cas9 in multiple DNA-bound states provide insights on the mechanism of Cas9 activation by target DNA.

Crystal structure of the RNA-guided endonuclease Cas9 bound to a guide RNA and a target DNA duplex reveals how base-specific recognition of a short motif known as PAM in the DNA target results in localized strand separation in the DNA immediately upstream of the PAM, allowing the target DNA strand to hybridize to the guide RNA.

By using cryo-EM and biochemical assays, Loeff et al. reveal the mechanistic basis for the protective function of the oxidoreductase PYROXD1 that maintains the activity of the tRNA ligase complex under aerobic conditions.

Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic oligoadenylates that activate effectors such as Csm6 ribonucleases. Here, Garcia-Doval et al. show that Enteroccocus italicus Csm6 degrades its cyclic hexa-AMP activator, and report the crystal structure of the protein bound to an activator mimic.

In this Review, Wiedenheft and colleagues retrace events that led from early endeavours to understand the role of Cas9 in CRISPR-mediated adaptive immunity to current efforts aimed at developing this enzyme for programmable genetic editing.

Compared to traditional Cas9 nucleases prime editors (PEs) are less active. Here the authors use OrthoRep, a yeast-based platform for directed protein evolution to enhance the editing efficiency of PEs: they identify mutations that have a positive effect on kinetics and use this knowledge to generate an efficient in vivo PE.

CRISPR-associated protein Csm6 is activated by a cyclic oligoadenylate second messenger generated by Cas10 activity in the CRISPR type III interference complex, representing a novel mechanism of CRISPR interference.

Eric Aird et al. present a strategy to increase the efficiency of homology-directed repair in CRISPR/Cas9-mediated genome editing. They show that tethering a single-stranded oligodeoxynucleotide to the ribonucleoprotein complex using a fused HUH endonuclease increases editing efficiency by up to 30-fold.

Structural studies on Scytonema hofmanni CRISPR-associated transposon protein complexes indicate a mechanism for RNA-guided DNA transposition involving Cas12k, TnsC and TnsB.

CRISPR enzymes require a defined protospacer adjacent motif (PAM) which can be limiting for editing applications. Here the authors recombine the PAM-interacting domain of SpRY with the N-terminus of Sc + + to generate a chimeric enzyme with highly flexible PAM preference: SpRYc.

Eukaryotic Argonaute proteins participate in RNA-guided RNA silencing pathways and are divided into AGO and PIWI clades, with functional and mechanistic differences. Here, the authors show that a deep-branching PIWI protein from Asgard archaea (the closest prokaryotic relatives of eukaryotes) displays hybrid features and may reflect an ancestral molecular architecture that preceded the divergence of eukaryotic AGOs and PIWIs.

A detailed understanding of the catalytic target DNA cleavage mechanism by CRISPR–Cas9 has been lacking. Now the key modulating role of Mg²⁺ in the conformational activation of Cas9 and the influence of active site residues on the protonation state of catalytic H840 are demonstrated, ultimately unravelling the catalytic mechanism.

SCF–FBXO31 scans proteins for C-terminal amidation and marks them for subsequent proteasomal degradation.

CRISPR-Cas12a is a powerful RNA-guided genome-editing system. Saha et al. show that an alpha-helical lid plays the central role in guiding the target DNA toward the single RuvC nuclease domain, resulting in a double-stranded DNA break.

Base editors are effective and safe for cholesterol reduction in non-human primates.

Cas9 remains bound to DNA after cleavage and its removal is required for DNA double-strand break repair. Here, the authors show that the HLTF translocase disrupts the Cas9- DNA post-cleavage complexes in a process that requires the HLTF HIRAN domain and ATPase activity.

The cryo-EM structure of the human core CPSF complex, containing CPSF160, WDR33, CPSF30 and Fip1 subunits, bound to its RNA target reveals the mechanism of PAS recognition.

GW182 has been implicated in the effector steps of microRNA-mediated repression and recently shown to interact with the Poly(A) binding protein C-terminal domain (PABPC1). The structure of PABPC1 in complex with a human GW182 paralog peptide, now gives insight into the interactions needed to elicit target deadenylation.

Through directed evolution, the PAM compatibility of the compact Cas9 variant CjCas9 was increased. Evolved CjCas9 shows higher nuclease activity at canonical and non-canonical sites and enables robust in vivo gene editing from single AAV vectors.

During mitosis, mitochondria partition into daughter cells through microtubule-based transport. Here the authors show that the mitochondrial protein Miro and the cytoskeletal-associated protein Cenp-F interact in a cell-cycle dependent manner to promote microtubule-directed movement of mitochondria.

The E3 ligase Hakai can interact with the m⁶A methylation machinery but its function is still unclear. Here, the authors show that Hakai is a conserved component of the m⁶A methyltransferase complex and provide functional and molecular insights into its role in regulating m⁶A levels in Drosophila.

In bacteria and archaea, clustered regularly interspaced short palindromic repeat (CRISPR) loci are unique pieces of foreign DNA (spacers) separated by repetitive sequences specific to the host. They provide an adaptive immune system against bacteriophages and plasmids. CRISPR loci are transcribed and processed by three endonucleases to produce short RNAs (crRNAs). Structures of Cse3-type endonuclease bound to its cognate repetitive RNA shows an RNA-induced conformational change in enzyme that aligns the RNA for site-specific cleavage.

This study defines how a short DNA sequence, known as the PAM, is critical for target DNA interrogation by the CRISPR-associated enzyme Cas9 — DNA melting and heteroduplex formation initiate near the PAM and extend directionally through the remaining target sequence, and the PAM is also required to activate the catalytic activity of Cas9.

The RECQL5 DNA helicase interacts with RNA polymerase II (Pol II) and inhibits transcription through an unknown mechanism. A series of structural and functional analyses now reveals that RECQL5 sterically blocks elongation while also competing with elongation factor TFIIS for binding to Pol II, suggesting a dual mode of transcriptional repression.

After transcription and processing, transfer RNAs must be exported from the nucleus to the cytoplasm, where translation occurs. This process is mediated by a dedicated nucleo-cytoplasmic transport factor called Xpot. Here, the structure of Schizosaccharomyces pombe Xpot is reported, unbound and in complex with both tRNA and another factor required for transport, RanGTP.

Simultaneous editing of the NCF1 and its pseudogenes in p47^phox-deficient chronic granulomatous disease is associated with homologous recombination and chromosomal rearrangements due to presence of multiple targets of high sequence similarity.

Prokaryotic Argonaute proteins, homologues of eukaryotic Argonaute proteins involved in RNA interference, have recently been demonstrated to mediate host defence in archaea and bacteria. In this Progress article, van der Oost and colleagues explore the structures and biological functions of the prokaryotic Argonaute proteins, and discuss their potential applications in genome editing.