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Nagahata, Kato and Yamada et al. provide cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases—HfmIscB, TbaIscB, YnpsCas9 and NbaCas9—each in complex with its guide RNA and target DNA, providing insights into CRISPR–Cas9 evolution.

The Retron-Eco7 is a genetic element involved in anti-phage defense that encodes two effector proteins (PtuA and PtuB) and cleaves the host tRNA. Here, the authors solved the Retron-Eco7 complex structure using cryo-EM.

The engineered Streptococcus pyogenes Cas9 variant SpCas9-NGv1, known to recognize relaxed NG protospacer adjacent motifs (PAMs) in human cells, can also mediate targeted mutagenesis with NG PAMs in rice and Arabidopsis. When fused with cytidine deaminase, it mediates C-to-T substitutions.

Ecto-nucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) is a type II transmembrane glycoprotein that hydrolyzes both ATP and cGAMP. Here the authors present the crystal structures of the extracellular domain of mouse ENPP1 in complex with 3′3′-cGAMP and the reaction intermediate pA(3′,5′)pG and discuss mechanistic implications.

Sperm-egg fusion requires the interaction between IZUMO1 on the sperm and JUNO on the egg. Here, the authors report the crystal structure of mouse JUNO, and use it to explain its lack of binding to folate, along with in vivofunctional analyses.

Hiramoto et al. develop a base-editing approach using SpCas9-NG, an engineered Cas9 with broad PAM flexibility, to correct a causative mutation in hemophilia B. Their approach is used to repair the point mutation in patient-derived iPSCs and restore coagulation factor IX expression in HEK293 cells and knock-in mice.

Francisella novicida Cas9 (FnCas9) has low cellular editing ability which limits its therapeutic utilities. Here, the authors rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants with high on-target editing efficiency, high precision, broadened target range, and flexible base editing outcomes.

A study of retrotransposon activity repurposes a retroelement called R2Tocc to create a programmable system called STITCHR that enables diverse genome edits including efficient, scarless large payload insertions.

Here Ishikawa et al. determine the cryo-electron microscopy structures of the PspCas13b–guide RNA complex alone and in complex with target RNA, as well as that of the dPspCas13b–ADAR2–guide RNA–target RNA complex, thereby providing mechanistic insights into RNA cleavage and editing.

CRISPR RNA-guided endonuclease Cas9 recognizes and cleaves the double-stranded DNA complementary to the RNA guide. Here the authors use high-speed atomic force micropcopy (HS-AFM) to visualize the conformational dynamics of Cas9 during its DNA targeting and cleavage processes.

Zucchini has been identified as an endoribonuclease responsible for the maturation of small RNA molecules that protect the genome from the damaging effects of unrestrained expression of mobile elements.

Using cryo-electron microscopy, the structural mechanism by which non-coding bridge RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination is explored.

A bispecific non-coding RNA expressed by the IS110 family of mobile genetic elements forms the basis of a programmable genome-editing system that enables the insertion, excision or inversion of specific target DNA sequences.

SpCas9 is a versatile genome-editing tool, but it is large and cannot be packaged efficiently in a AAV vector, limiting its application. The reported engineered Campylobacter jejuni Cas9 variant exhibits enhanced cleavage activity and a broader targeting range, expanding the CRISPR-Cas toolbox for therapeutic genome engineering.

Cryo-electron microscopy structures of Drosophila Dicer-2–R2D2 complexes with and without small interfering RNA reveal how the RNA is presented to Argonaute in the correct orientation for viral gene silencing.

The cryo-EM structure of the IscB–ωRNA–target DNA complex provide insights into the ωRNA-guided DNA cleavage mechanism by IscB and the evolution of the type II CRISPR-Cas9 effector complexes

While prime editing is a promising technology, PE2 systems often have low efficiency. Here the authors fuse a Rad51 DNA-binding domain to create hyPE2 with improved editing efficiency.

The RNA-guided DNA endonuclease Cas9 from Corynebacterium diphtheriae (CdCas9) recognizes a promiscuous protospacer adjacent motif (PAM). Here the authors provide insights into the CdCas9-mediated PAM recognition mechanism by determining the 2.9 Å crystal structure of CdCas9 in complex with the guide RNA and its target DNA, which is of interest for engineering of CRISPR-Cas9 genome-editor nucleases.

PIWI-clade Argonautes bind PIWI-interacting RNAs (piRNAs) to silence transposons and maintain genome integrity in animal gonads. Here the authors present the crystal structure of a Drosophila Piwi–piRNA complex isolated from cultured fly ovarian somatic cells and find that it contains a non-canonical DVDK tetrad, and lacks slicer activity in vitro.

The TDF peptide interacts with the leucine-rich repeat receptor-like kinase TDR to regulate meristem differentiation in plants. Here, the authors solve the structure of the extracellular domain of TDR in complex with TDIF and propose a mechanism for TDIF recognition.

:Autotaxin (ATX) is a secreted enzyme that produces lysophosphatidic acid (LPA), which in turn activates GPCRs to elicit cellular responses. The crystal structures of mouse ATX in its apo state and bound to different LPAs and functional work reveal a hydrophobic tunnel that could participate in direct delivery of the product to its cognate GPCRs.

The CRISPR-Cas9 system, a powerful tool for genome editing, has been engineered to activate endogenous gene transcription specifically and potently on a genome-wide scale and applied to a large-scale gain-of-function screen for studying melanoma drug resistance.

T helper 2 (Th2) cells and type 2 innate lymphoid cells (ILC2) respond differently to interleukin-33 (IL-33) stimulation. Here the authors show that a phosphatase, Dusp10, is expressed in Th2, but not ILC2, to dephosphorylate p38 kinase, reduce GATA3 transcription factor activity, and suppress the induction of IL-5 in response to IL-33.

SELEX selections and crystallographic analyses have allowed development of a DNA aptamer that inhibits autotaxin with high potency and specificity, and exhibits efficacy against bleomycin-induced pulmonary fibrosis in model mice.

Base editors that modify both adenosine and cytosine broaden the potential applications of base editing.

An automated amplification-free digital RNA detection platform using CRISPR-Cas13a and magnetic bead technology implemented in a microchamber device demonstrates sensitive SARS-CoV-2 genomic RNA detection and variant discrimination.

The targeting range of the CRISPR endonuclease Cpf1 is increased three-fold by molecular engineering.

A crystal structure of Brevibacillus laterosporus Cas9 highlights the mechanistic diversity among the CRISPR-Cas9 effector enzymes.

The authors develop a platform (SATORI) that enables accurate and rapid detection of single-stranded RNA at a single-molecule level without a pre-amplification step. As a proof-of-concept, they demonstrate its utility in detecting the SARS-CoV-2 N gene at a minimum concentration of ~5 fM, which is much lower than other amplification-free CRISPR-Cas-based methods.

Seiya Oura and Taichi Noda et al. overcome the challenge of gene editing in CAG repeats, such as those causing Huntington’s Disease, using their recently developed SpCas9-NG variant. They demonstrate that SpCas9-NG can precisely edit and contract the CAG repeat tracks in a Huntington’s Disease mouse model, opening new avenues for research in this disease.