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Recent advances in genome editing technologies have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Programmable nucleases, particularly the CRISPR/Cas system, are revolutionizing our ability to interrogate the function of the genome and can potentially be used clinically to correct or introduce genetic mutations to treat diseases that are refractory to traditional therapies. This collection of recent articles from the Nature Research journals provides an overview of current progress in developing targeted genome editing technologies. A selection of protocols for using and adapting these tools in your own lab is also included.
The authors introduce MACHETE (molecular alteration of chromosomes with engineered tandem elements), a clustered regularly interspaced short palindromic repeats directed Cas9-based system for the efficient deletion of megabase-sized genomic regions.
This protocol provides extensive guidelines and detailed steps to generate novel bio-engineered bacterial strains using CRISPR-associated transposase (CAST) systems, with available plasmids and standard molecular biology techniques.
The authors provide an expanded CRISPR–Cas9-assisted recombineering toolkit for rapid and efficient engineering of genetically intractable Pseudomonas aeruginosa isolates.
The authors describe a new base editing system—the transformer base editor—to induce efficient editing with no observable genome-wide or transcriptome-wide off-target mutations, both in mammalian cells and in mice.
This protocol for universal and proficient Pseudomonas recombineering uses phage-encoded homologous recombination–Cas3 systems, including SacB counterselection and Cre site-specific recombinase for two- or three-step seamless genome modification.
The authors provide a protocol for cytosine base editing to introduce precise substitutions into the genome of zebrafish, an important model for genetic studies and in vivo disease modeling.
Detect-seq (dU-detection enabled by C-to-T transition during sequencing) enables off-target evaluation of programmable cytosine base editors. DNA is chemically labeled, and the editing intermediate dU is enriched for sequencing by biotin pull-down.
This CRISPR-Combo system enables efficient multiplexed orthogonal genome editing and transcriptional activation in plants. Here, the use of CRISPR-Combo is demonstrated for speed breeding of transgene-free, genome-edited Arabidopsis and enhancing rice regeneration with more germline mutations.
This protocol for Multiplexed Intermixed CRISPR Droplets uses microfluidics to create droplets containing Cas9, multiplexed single-guide RNAs and corresponding DNA barcodes, allowing large-scale genetic screens to be performed in F0 zebrafish.
BEAN is a Bayesian approach for analyzing base editing screens with improved effect size quantification and variant classification. Applied to low-density lipoprotein (LDL)-associated common variants and saturation base editing of LDLR, BEAN identifies new LDL uptake genes and offers insights into variant structure–pathogenicity mechanisms.
Experiments in mice show that designed epigenome editors that contain domains of transcriptional repressors can enable stable epigenetic silencing of Pcsk9, a gene with a role in cholesterol homeostasis, without inducing DNA breaks.
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.
The lack of control over Cas13 activity has limited its utility. Here the authors report Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands (CRISTAL), controlled by orthogonal split inducible Cas13 effectors that can be turned ON or OFF, providing precise temporal control.
Huang, Qin, Shang et al. profile double-strand breaks (DSBs) generated by C-to-G base editors (CGBEs) and find that HMCES protects abasic sites and reduces CGBE-triggered DSBs.
Massive-scale mutational screening across 385 genes reveals a wide spectrum of alleles that govern tunable T cell functions, including cytokine production and cytotoxicity.
Exogenous control of genes in vivo is important. Here the authors report a system that can be inducibly activated through thermal energy produced by ultrasound absorption and use this to control induction of gene activation and base editing: they apply this in cell lines and in a mouse model.
The deaminases of base editors alone can induce unpredictable off-target edits. This paper describes a simple and generalizable solution for base editors containing distinct deaminases to suppress off-target edits in plant, yeast and human cells.
Assembloids are integrated with CRISPR screening to investigate the involvement of 425 neurodevelopmental disorder genes in human interneuron development, showing endoplasmic reticulum displacement before nuclear translocation and interference from LNPK deletion, resulting in abnormal migration.
An in vivo single-cell CRISPR screening method identifies transcriptional phenotypes of 22q11.2 deletion syndrome associated with a broad dysregulation of a class of disease susceptibility genes that are important for RNA processing and synaptic function.
Huang et al. develop a potent genome editing toolkit to generate transgene-free genome-edited plants in the T0 generation by co-editing of ALS gene (without obvious fitness costs) and gene(s) of interest via Agrobacterium-mediated transient expression.
During transformation via Agrobacterium tumefaciens, sequence composition of T-DNA (that is, DNA repeats) affects T-DNA concatenation in Arabidopsis. In addition, loss of specific DNA repair proteins dramatically decreases T-DNA concatenation levels. This study showcases the potential of T-DNA concatenation as a tool to enhance gene editing.
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.
A CRISPR–Cas12a promoter editing system enabled efficient engineering of quantitative trait variations of grain starch and seed size in rice, and achieved an innovative Green Revolution trait through editing of the OsD18 promoter.
Adenine base editing successfully corrected a MYH7 pathogenic variant that causes hypertrophic cardiomyopathy in human cardiomyocytes and a mouse model of the disease, highlighting the potential of the approach to correct monogenic variants causing cardiac disease.
The CRISPR–Cas9 system is widely studied for its role as a phage defence system and for gene editing applications, but its evolutionary origins are poorly understood. Here the authors use ancestral sequence reconstruction to determine the evolutionary history and ancient protein sequences of Streptococcus pyogenes Cas9 ancestors.
Targeted genome modification using CRISPR–Cas genome editing, base editing or prime editing is driving base research in plants and precise molecular breeding. The authors review the technological principles underlying these methods, approaches for their delivery in plants, and emerging crop-breeding strategies based on targeted genome modification.
L. Villiger, J. Joung et al. review CRISPR applications for programmable editing of the genome, epigenome and transcriptome. They discuss how CRISPR–Cas systems can be optimized to further improve editing specificity and efficiency and highlight a multitude of applications in basic biological research and for changing clinical practice.
In this Tool of the Trade article, Nicolas Mathey-Andrews describes the generation and use of a prime editor mouse that enables in vivo modelling of the multitude of cancer alleles found in human tumours.
CRISPR-based genome editing has the potential to treat many human genetic diseases, but achieving stable, efficient and safe in vivo delivery remains a challenge. This Review assesses current delivery systems for genome editors—focusing on adeno-associated viruses and lipid nanoparticles—and highlights data from recent clinical trials. Emerging delivery systems and ongoing challenges in the field are discussed.
CRISPR-based genetic screens are providing new insights into the consequences of deficiencies in DNA damage response and repair pathways. These include insights into the regulation of homologous recombination and of replication stress and their crosstalk with other repair pathways, into novel cancer therapies and into the basis of cancer-drug resistance.
In this Review, Chen and Liu discuss the latest developments in prime editing systems, including improvements to their editing efficiency and capabilities, as well as diverse emerging applications in research and preclinical therapeutic studies.
MEGA is a new CRISPR-based RNA-editing platform with the ability to enhance the fitness of CAR T cells; it may also overcome certain limitations of conventional DNA-targeting CRISPR–Cas9 systems.
Patients who received an in vivo CRISPR-based gene-editing therapy for hereditary angioedema showed no serious side effects and nearly complete disease control.
Base editors can restore the expression of survival motor neuron protein to therapeutically beneficial levels in animal and cell models of spinal muscular atrophy.
A major challenge in human genetics is the prioritization of causal genes in common complex diseases. A genome-wide CRISPR screen for intracellular insulin content in a human β-cell line has now identified a new candidate gene for type 2 diabetes, demonstrating the utility of this screening approach in β-cells.
CRISPR cell and gene therapy have been designed largely with respect to a single reference human genome. A new study reveals how human genetic diversity could lead to off-target effects and presents a new tool to identify these risks.