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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.
This protocol describes prime editing (PE) and twinPE experiments as well as the design and optimization of pegRNAs. The authors provide guidelines for selecting the proper PE system for a given application and how to perform PE in mammalian cells.
This protocol for base editing in cultured mammalian cells provides guidelines for choosing target sites, appropriate base editor variants and delivery strategies, as well as detailing the computational analysis of base-editing outcomes using CRISPResso2.
GUIDE-seq (genome-wide unbiased identification of double-stranded breaks enabled by sequencing) is a sensitive, unbiased, genome-wide method for defining the specificity of genome-editing nucleases in living cells.
Bacterial genome-wide gene fitness is assessed by CRISPRi-seq. The procedure includes a pipeline for single-guide RNA library design, workflows for pooled CRISPRi library construction, growth assays, sequencing and read analysis fitness quantification.
The authors present a protocol for using the CRISPR–Cas9 genome editing system to knock out a gene of interest in human intestinal tissue–derived enteroids by lentiviral transduction and single-cell cloning.
This protocol uses PlantPegDesigner to design and optimize prime editing guide RNA and engineered plant prime editor vectors for efficient prime editing in monocot plants.
The authors provide a versatile gene therapy approach that is mutation- and gene size–independent, using dCas9-VPR–based transcriptional activation of functionally equivalent genes. They show how to apply this for gene therapy for inherited retinal dystrophies in mice as an example.
The authors provide protocols for rapid and scalable genome engineering in somatic cells of the liver and pancreas through both viral and nonviral delivery of CRISPR components into living mice.
This chromosome-engineering protocol generates heritable chromosomal rearrangements in A. thaliana; by combining SaCas9 with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements can be made and homozygous lines can be established.
Tamlo-R32, an engineered wheat mutant allele of the Mildew resistance locus O (MLO) gene, confers resistance to powdery mildew, retains robust wheat growth, and can be transferred to other agriculturally important wheat varieties.
A first-in-human phase I clinical trial demonstrates the feasibility and safety of non-viral precision genome-engineering of a personalized adoptive cell transfer anticancer therapeutic.
Adeno-associated viruses with size-optimized genomes encoding compact adenine base editors enable therapeutic base editing in mice at low doses and high editing efficiencies.
The delivery of oligonucleotides disrupting the secondary structure of single-guide RNAs and of short interfering RNAs targeting Cas9 mRNA can enhance lipid-nanoparticle-mediated gene editing in vivo in tissues other than the liver.
Brain-wide Cas9-mediated cleavage of a disease-causing gene after a single intravenous or intrahippocampal injection alleviates amyloid-beta-associated pathologies in mouse models of familial Alzheimer’s disease.
Leng et al. establish CRISPRi screens in astrocytes to dissect pathways controlling inflammatory reactivity. They uncover two distinct inflammatory reactive signatures that are inversely regulated by STAT3 and validate that these exist in human disease.
Dräger et al. establish a rapid, scalable platform for iPSC-derived microglia. CRISPRi/a screens uncover roles of disease-associated genes in phagocytosis, and regulators of disease-relevant microglial states that can be targeted pharmacologically.
Tian et al. conducted a genome-wide CRISPRi/CRISPRa screen in human neurons and uncovered a neuron-specific link among prosaposin, lipofuscin and ferroptosis. The CRISPRbrain data commons enables comparison of gene function across human cell types.
Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. Here the authors present PepSEq, a high-throughput method for screening a large library of peptides that influence prime editing efficiency.
Toxicity of CRISPR/Cas9 induced DNA breaks depends on their repair mechanism, and on the chromatin environment at the cut site. Here the authors show that edits in active genes or regulatory elements can incur a higher toxicity via a TP53-dependent mechanism.
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.
Base editing is promising for gene therapy, but in vivo delivery has been limiting. Here the authors perform structure-based rational engineering of the cytosine base editing system Target-AID to minimise off-target effects and decrease its size.
CRISPR-Cas12f nucleases can be effectively packaged into AAVs for gene therapy, but a systematic evaluation of editing outcomes is lacking. Here the authors perform a comprehensive assessment of 4 Cas12f proteins and compare to Cas9 and two Cas12a proteins at a number of sites.
The success of CRISPR experiments relies on the choice of gRNA. Here the authors report crisprVerse, which enables efficient gRNA design and annotation for methods including CRISPRko, CRISPRa, CRISPRi, CRISPRbe and CRISPRkd, enabled for RNA- and DNA-targeting nucleases, including Cas9, Cas12 and Cas13.
Pan et al. develop a versatile CRISPR-Combo platform for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants, representing a versatile genome engineering tool with promising applications in crop breeding.
A method for targeted mutagenesis of mitochondrial genomes is presented. It combines site-specific DNA cleavage with selection for mutations that confer cleavage resistance, and produces genetically stable plants with edited mitochondrial genomes.
Manipulation of genetic exchange is an important objective of plant breeders. Using chromosome engineering to invert a 17.1 Mb fragment on chromosome 2 in Arabidopsis thaliana, meiotic recombination could be suppressed in nearly the entire chromosome.
Plant-optimized transcription activator-like effector-linked deaminases enable site-specific A-to-G base editing in the chloroplast genome, leading to heritable homoplasmic base conversions and phenotypic changes.
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.
This Review discusses strategies for the genetic engineering of adoptive T cell immunotherapies with a focus on approaches harnessing transgenic T cell receptors or chimeric antigen receptors to treat cancer. The authors also discuss the more complex levels of genetic regulation that will be needed to ensure both safety and efficacy.
In this Review, Brouns and colleagues discuss our current understanding of RNA-targeting type III and type VI CRISPR–Cas systems by detailing their composition, properties and defence processes, and describing the biological rationale behind the broad activated immune responses as an effective strategy to combat viral infection.
CRISPR–Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, Wang, Pausch and Doudna examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR–Cas immune systems and deployed for wide-ranging genome editing applications.
Gene-based therapies offer the promise of long-lasting clinical benefit for both genetic and sporadic neurodegenerative diseases. Sun and Roy highlight recent successes and caveats, offering a prospective glimpse into this rapidly emerging arena.
This Review summarizes current technologies for organellar genome editing in plants and their applications, and highlights opportunities brought by emerging techniques such as RNA editing, prime editing and new transformation methods.
Rubin et al. report the development of a programmable organism- and locus-specific genome editing approach that can target microorganisms in their native community context, without the need for isolation.
A study in Nature describes ‘DNA Typewriter’, a prime-editing-based DNA recording technology that can capture the order of large numbers of distinct molecular events in mammalian cells.
Same Cas9 protein, two different jobs: the CRISPR-Combo genome engineering strategy enables simultaneous gene activation and genome editing for different targets through changes to the guide RNA structure.