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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.

Existing TadA-derived CBEs exhibit residual A•T-to-G•C editing activity and suffer from lower activity at several sequence contexts and with non-SpCas9 targeting domains. Here, the authors use phage-assisted evolution to evolve CBE6 variants that address these limitations.

Exo-PE is an approach to improve prime editing efficacy for insertions while maintaining precision.

A key step for the use of CRISPR-Cas9 in any study is the design of the guide RNA, however the underlying principles are still poorly understood. Here the authors show that multiple protospacer adjacent motif sequences are refractory to efficient targeting and repair.

Cas9 RNA-guided engineered nucleases (RGENs) induce site-specific DNA cleavages in cultured cells and organisms and are used widely as genome-editing tools. Here, the authors develop an RGEN-based technology to genotype both RGEN-induced mutations and cancer-associated mutations in human cell lines.

The microbial RNA-guided CRISPR/Cas9 system has robust genome-editing activities, but the off-target effects of the Cas9 nuclease have only recently begun to be analysed. Here the authors provide evidence for high specificity of the Cas9 nuclease on targeting of the Tafazzin gene in human-induced pluripotent stem cells and demonstrate the impact of single-nucleotide variations of the human genome on Cas9 specificity.

Genomic rearrangements have important functional consequences for cancer. Here, Choi and Meyerson use CRISPR/Cas technology to generate translocations and inversions that are known drivers of lung cancer, and demonstrate the utility of this technology for studying the role of genomic rearrangements in disease.

The fission yeast, Schizosaccharomyces pombe, is a valuable model organism, but the lack of a portable RNA Pol III promoter has prevented the implementation of the CRISPR/Cas9 system. Here the authors develop a CRISPR/Cas9 system that achieves selection-free specific mutagenesis with very high efficiencies in S. pombe.

CRISPR-Cas9 is a powerful tool for genome editing; however, difficulties in generating pools of paired guide RNAs limit its applicability to large-scale screening experiments. Here the authors report a one-step method for rapid and efficient generation of pooled libraries of guide RNA pairs.

Tools derived from synthetic biology offer powerful means to refine drug delivery and disease detection. Liu et al. engineer a logical AND gate using CRISPR-Cas9 to drive gene expression only cells in which two promoters are active, and use it to selectively inhibit the growth of bladder cancer cells in vitro.

A photoconvertible reporter of the ubiquitin-proteasome system permits detection of its activity independent of protein synthesis and is applied to study cell type– and age-specific protein degradation in living Caenorhabditis elegans.

Transcription activator–like effectors are used for in vivo activation and repression of endogenous promoters and enhancers in the fruit fly.

Culex mosquitoes are carriers of major diseases like West Nile virus and are a public health concern. Here the authors present a CRISPR-Cas9 gene drive as a control technology in the Culex quinquefasciatus mosquito species.

Hemimetabolous insects comprise many pests but introducing targeted mutations into these species has been difficult. This paper reports efficient targeted mutagenesis, and the generation of homozygous knockouts, in crickets based on zinc finger nucleases or transcription activator-like effector nucleases.

In addition to causing A-to-G base transitions, adenine base editors also cause C base substitutions

Ddd-Aderived cytosine base editors (DdCBEs) are important for research of mitochondrial DNA mutation diseases. Here the authors report a strategy for screening and characterising dsDNA cytidine deaminases, and identify 7 DddA homologs which they optimise to minimise nuclear and mitochondrial off-target editing.

The safety of CRISPR-Cas9 editing is a concern. Here the authors use whole genomic analysis by 10x linked-read sequencing and optical genome mapping to interrogate the genome integrity after editing: they see large structural variants at on-target sites and unexpected large chromosomal deletions.

Cas9 is a powerful genome-manipulation enzyme, although how its catalytic activity is controlled is not completely solved. Now, cryo-electron microscopy structures of Acidothermus cellulolyticus Cas9 provide atomic-level insights into its activation involving DNA binding, conformational changes and the formation of its two active sites.

Engineered zinc finger nucleases achieve 98% knockout of a target gene in human T cells without detectable off-target activity.

Cpf1 is a promising alternative to Cas9 though indel generation efficiency is target dependent. Here the authors show that the addition of a polyU 3′ overhang can improve the efficiency of low efficiency guide RNAs.

TadA deaminases widely used in many base editors lack post-translational control in cells. Here the authors report a split adenine base editor (sABE) using chemically induced dimerisation (CID) to control the catalytic activity of TadA8e and show this can be used for PCSK9 gene editing in the mouse liver.

Adenine base editing is used to treat a mouse model of Duchenne muscular dystrophy and to create defined mutations in mouse embryos.

The large size of nucleases of commonly used CRISPR-Cas systems reduces the delivery efficiency of these gene-editing tools for therapeutic applications. Now, the cryo-electron microscopy structure of the miniature Acidibacillus sulfuroxidans Cas12f1 reveals the determinants for its activity despite its small size.

Off-target cleavage by CAS9 or TALEN genome editing tools is detected by integrase-defective lentiviral vectors.

Gene targeting via homologous recombination is achieved in the zebrafish with TALENs and double-stranded DNA donors, expanding the range of experimental possibilities in this organism.

Limited work has been done on concurrent C-to-G and A-to-G base editing. Here the authors test how a number of chromatin-associated factors affect base editing and show that HMGN1 enhanced the efficiency; by fusing HMGN1 to GBE and ABE they develop a CRISPR-based dual-function A-to-G and C-to-G base editor (GGBE).

Advances in genome sequencing, editing and synthetic biology have enhanced the feasibility of large-scale genome engineering, termed genome writing. In this Opinion article, Chari and Church discuss the strengths and limitations of diverse strategies for genome writing, including extensively modifying existing genomes versus synthesizing genomesde novo, and they provide future visions for writing large genomes.

HDRobust is a high-precision tool for homology-directed repair-mediated single-nucleotide editing.

Programmable nucleases — including ZFNs, TALENs and RGENs derived from the prokaryotic CRISPR–Cas system — enable targeted high-precision genome engineering. This Review summarizes current knowledge of nuclease-specific features, individual pros and cons, and approaches to improve the efficiency of these enzymes to help researchers to choose the most appropriate tool for various genome editing experiments and applications.

The widely used SpCas9-NG tool can edit genomic targets, but unfortunately also the guide RNA sequences that are present in the transfer DNA and integrated into the plant genome. The authors propose a strategy to alleviate this self-editing risk.

The generation of CRISPR-mediated transcriptional activation (CRISPRa)-competent cell lines pose significant technical challenges. Here the authors report a platform for production of CRISPRa-ready cell populations which they combine with optimised expressed and synthetic gRNA scaffolds to enhance functionality.

Application of CRISPR-Cas13d is limited by the inability to predict on- and off-targets. Here the authors perform CRISPR-Cas13d proliferation screens followed by modeling of Cas13d on- and off-targets; they design a deep learning model, DeepCas13, to predict the on-target activity of a gRNA.

A study developed genomic resources and efficient transformation in the orphan crop groundcherry, and managed to improve productivity traits by editing the orthologues of tomato domestication and improvement genes using CRISPR–Cas9.

Properties of cytidine and adenosine deaminases lead to off-target effects for cytosine base editors (CBEs) and adenine base editors (ABEs). Here the authors report that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single/double mutations with minimised off-targets.

Methods to predict genome-wide off-target activities of prime editors (PEs) are currently lacking. Here the authors report a cell-based assay, TAgmentation of Prime Editor sequencing (TAPE-seq), that provides genome-wide off-target candidates for PEs.

Hypercompact CRISPR-Cas12f systems have been engineered to generate miniABEs but these have limitations. Here the authors generate Cas12f-derived miniCBEs and develop miniABEs with improved editing and targeting scopes; they use these to correct pathogenic mutations in cell lines and introduce mutations in vivo.

Visualisation of point mutations in situ is informative for studying genetic diseases. Here the authors report single guide genome oligopaint via local denaturation fluorescence in situ hybridisation, sgGOLDFISH, a direct hybridisation genome imaging method with single-nucleotide sensitivity.

Strategies to improve the specificity of nuclease-based prime editor (PEn) are needed. Here the authors report a 53BP1-inhibitory ubiquitin variant-assisted PEn platform (uPEn) to inhibit NHEJ and enable precise prime editing for generation of insertions, deletions and replacements.

The best base editor for specific applications is predicted with a deep learning model.

Increased-fidelity SpCas9 variants have been developed, but often show proportionally reduced activity. Here the authors characterise the on-target activity and off-target propensity of SuperFi-Cas9 in mammalian cells and also see strongly reduced activity but with high fidelity features.

A large number of cell disease models with pathogenic SNVs are needed. Here the authors report an automated high-throughput platform to perform the genome editing process from gRNA design to the analysis of the editing results; they characterise in situ base editing outcomes.

The activity of standard Cas9-based genome-editing systems can be constrained by the addition of cytosine stretches to the 5′-end of conventional single-guide RNAs.

Prime editing can efficiently correct the sickle-cell allele to produce wild-type haemoglobin in patient haematopoietic stem cells that engraft efficiently in mice, yielding erythrocytes resistant to hypoxia-induced sickling.

The study describes the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in plants by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components.

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.

Specific RNAs are detected in live cells by harnessing endogenous ADAR enzymes.

Existing methods for generating sgRNA predictions do not account for the tracrRNA sequence. Here the authors report an on-target model, Rule Set 3, to generate optimal predictions for multiple tracrRNA variants, and validate this on a new dataset of sgRNAs showing improvement over prior prediction models.

CRISPR-Cas induced HDR methods tend to have a low efficiency. Here the authors report an HDR improvement strategy, Recursive Editing, that selectively retargets undesired indel outcomes to create additional opportunities for HDR; they introduce REtarget, a tool for Recursive Editing experimental design.