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Distinguishing self from non-self is a vital function for immune systems to repel invaders without inducing autoimmunity. One system, which protects bacteria and archaea from invasion by phage and plasmid DNA, involves clustered, regularly interspaced, short palindromic repeat (CRISPR) loci. Here, in Staphylococcus epidermidis, the mechanism of CRISPR self/non-self discrimination is defined.

Nme2Cas9 has been well established as a genome editing platform. Here the authors engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors and validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

Resistance of gRNA to ubiquitous ribonucleases is required for CRISPR-Cas9-based therapeutics. Here, the authors explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor, and identify modified versions that are more potent and stable than their unmodified counterparts in editing human cells.

Long-term expression of Cas9 following precision genome editing in vivo may lead to undesirable consequences. Here we show that a single-vector, self-inactivating AAV system containing Cas9 nuclease, guide, and DNA donor can use homology-directed repair to correct disease mutations in vivo.

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.

Bacteria and archaea are microorganisms that often use RNA-guided defences called CRISPR to destroy the genomes of viruses that infect them. It now emerges that viruses make RNAs that act as mimics to divert such defences.

A split prime editor simplifies delivery.

Retrotransposons replicate their genetic information through target-primed reverse transcription (TPRT). Here the authors report a template-jumping prime editor (TJ-PE) to act similarly to TPRT and achieve insertions of large DNA fragments at endogenous sites: they rewrite a mutated exon in the mouse liver.

The CRISPR system uses small RNAs to provide bacteria and archaea with an adaptive defence mechanism against foreign nucleic acids. Recent studies have increased the understanding of mechanisms of CRISPR interference, its roles in microbial physiology and evolution and its potential applications.

Cpf1 is one of a growing number of class II CRISPR-Cas effectors that expand both our understanding of bacterial immunity and our genome-editing toolset.

DNA repair activity of reverse transcriptase may negatively affect prime editing precision.

A hybrid CRISPR–Cas9 variant is able to specifically and efficiently target a larger proportion of the genome.

Many bacteria and archaea protect themselves from viruses and other invasive genomes through a genetic interference pathway. The small RNAs that guide this defence specify the direct cleavage of foreign DNA. See Article p.67

CRISPR immunity begins with the acquisition of sequences from invading nucleic acids through spacer integration into a CRISPR locus. Off-target integration of spacers into other parts of the genome is now implicated as a spontaneous source of new CRISPR loci.

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.

Anti-CRISPR proteins offer the means of regulating CRISPR-Cas9 activity. Here the authors present the structure and biochemical characterisation of AcrIIC2_Nme alone and in complex with Cas9.

dCas9 fused to ascorbate peroxidase (APEX2) biotinylates proteins around targeted loci so they can be enriched and characterized.

Small RNAs can silence target genes by a variety of pathways and mechanisms. Specific microRNAs have now been shown to partition into distinct effector complexes, which may in turn affect their silencing output.

PE-tag is a genome-wide approach to map the off-target effects of in vitro and in vivo prime editing.

A DNA polymerase editing system offers advantages over prime editing.

Worldwide pandemics of obesity and diabetes prompt an urgent need for new approaches to their prevention and cure. Here the authors present a CRISPR-based strategy that enhances the therapeutic potential of human adipocytes when implanted in obese mice.

Protospacer adjacent motif (PAM) requirements limit the target range of CRISPR endonucleases. Here, the authors graft the 5\(^{\prime}\)-NAAN-3\(^{\prime}\) PAM-interacting domain of SmacCas9 onto SpyCas9 to create adenine dinucleotide targeting chimeras.

A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of ALS and FTD. Here, the authors demonstrate CRISPR/Cas9 excision of the expansion results in a rescue of disease mechanisms in vivo and in vitro.

Prime editors use a template sequence within their pegRNA to facilitate nucleotide substitutions or local indels. Here the authors use AAVs to deliver a split-intein prime editor in vivo to correct a pathogenic mutation.

In vivo assessment of nuclease off-target activity has primarily been indirect or through ChIP-based detection of double-strand break DNA repair factors, which can be cumbersome. Here, the authors show that GUIDE-tag, enables one-step off-target genome editing analysis in mouse liver and lung.

A conserved region of U6 small nuclear RNA has been implicated in binding an essential metal ion. Is this a glimpse of the spliceosome's catalytic core?

Prime editing efficiency is enhanced using a mutated reverse transcriptase and addition of deoxynucleosides.

Therapeutic genome engineering relies on the development of reliable, robust and versatile tools. Here the authors develop Cas9-Cas9 chimeras with high target site activity that generate predictable deletions.

The Hermansky-Pudlak Syndrome 4 protein (HPS4) mediates trafficking between late endosomes and lysosomes and is now shown to inhibit small RNA-mediated silencing (RNAi) in flies and human cells. Components of the ESCRT complex, which mediates late endosome trafficking, are required for efficient miRNA-mediated silencing and additional results support the idea that RNAi effectors are functionally linked to endosome-associated compartments.