Specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples.
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 already 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 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 computational protocol functionally links bacterial or archaeal genes within a dataset, enabling reliable functional predictions to be extracted for uncharacterized genes. As one example, the authors describe the ‘CRISPRicity’ metric to link genes to CRISPR–Cas systems.
Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD
This protocol describes procedures for generating genome-edited mouse models by injecting CRISPR reagents into oviducts of pregnant females and subsequently electroporating the reagents into zygotes in situ, thus bypassing cumbersome ex vivo handling of embryos.
This protocol describes how to create functional chromosome fusions in yeast through sequential rounds of CRISPR–Cas9-guided homologous recombination. Each round of pairwise chromosome end-to-end fusion deletes two telomeres and one centromere.
Here the authors describe CAPTURE, a versatile and sensitive protocol to detect spacer-acquisition events in native CRISPR arrays using nested PCR amplification and amplicon size selection.
Design, execution, and analysis of CRISPR–Cas9-based deletions and genetic interaction networks in the fungal pathogen Candida albicans
Here, the authors provide protocols for CRISPR–Cas9-based genetic manipulation of Candida albicans, using a gene-drive strategy that allows genetic interaction networks to be established for this important fungal pathogen.
MAGeCKFlute is an algorithm for the analysis and visualization of CRISPR screen data. Starting from sequencing reads and an sgRNA library, the algorithm normalizes results and represents them as pathway enrichment classifications.
A new DNA-editing technique called prime editing offers improved versatility and efficiency with reduced byproducts compared with existing techniques, and shows potential for correcting disease-associated mutations.
Chassis-independent recombinase-assisted genome engineering (CRAGE) enables the integration of plasmids encoding biosynthetic gene clusters into the chromosomes of diverse bacteria to optimize production of natural products in non-native strains.
CRISPR–Cas9 engineering of the Drosophila Atpα gene (encoding the α-subunit of the sodium pump) is used to study the ability of mutations that evolved independently in several insect orders to confer resistance to keystone plant toxins.
Selectable markers are widely used in cell engineering but there is only a limited variety to choose from. Here the authors split markers using inteins, allowing up to six transgene integration events to be selected for with one marker.
Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia
Delivering biological cargo to airway epithelial cells is very challenging. Here, the authors use engineered amphiphilic peptides to shuttle proteins and CRISPR RNPs into airway cells in vivo.
Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations
DNA repair by microhomology-mediated end joining creates precise deletions based on flanking microhomologies. Here the authors use CRISPR-Cas9 to recreate pathogenic deletion mutations using existing microhomologies in the human genome identified by their program MHcut.
Previous gene editing in haematopoietic stem cells (HSCs) has focussed on a heterogeneous CD34+ population. Here, the authors demonstrate high efficiency CRISPR/Cas9-based editing of purified long-term HSCs using non-homologous end joining and homology-directed repair, by directing isoform-specific expression of GATA1.
In vivo non-invasive monitoring of dystrophin correction in a new Duchenne muscular dystrophy reporter mouse
Dystrophin-deficient mice are used to test corrective strategies for Duchenne muscular dystrophy, but evaluation of dystrophin expression requires collection of tissue samples from specific muscles and time points. Here, the authors generate mice in which dystrophin expression is coupled to luciferase, and show that bioluminescence allows non-invasive monitoring of dystrophin expression following genome editing.
Tiling-sgRNA designs allow the in situ evaluation of protein domain functions. Here the authors present ProTiler - a computational method to predict CRISPR knockout hyper-sensitive regions, revealing previously unannotated domains.
In-depth characterization of adeno-associated virus (AAV)-mediated CRISPR delivery is still lacking. Here, the authors show high levels of integration into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in vivo.
Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways
DNA methylation plays an important role in regulating a wide variety of cellular processes and is implicated in a range of diseases. Here the authors present Casilio-ME to assemble protein complexes to demethylate target loci.
Application of highly specific Cas9 variants can be restricted by the design of the guide RNA. Here the authors present DeepHF, a gRNA activity prediction tool built from genome-scale screens of 50,000 guides covering 20,000 genes.
The role of CTCF-bound insulator elements in enhancer-gene interactions and transcriptional regulation remains poorly understood. Here, the authors investigate multiple epigenome editing strategies for perturbing individual CTCF-bound insulators, and evaluate their effects on genome topology and transcription.
Off-target effects in CRISPR screens for essential regulatory elements have not been systematically evaluated. Here the authors find Cas9 nuclease, CRISPRi/a each have distinct off-target effects, and that these can be accurately identified and removed using the GuideScan sgRNA specificity score.
Mucopolysaccharidosis type I (MPSI) is a lysosomal storage disease caused by insufficient iduronidase (IDUA) activity. Here, the authors use an ex vivo genome editing approach to overexpress IDUA in human hematopoietic stem and progenitor cells and show it can phenotypically correct MSPI in mouse model.
CRISPR activation strategies in bacteria are limited due to the reliance on σ70 promoters. Here the authors demonstrate eukaryote-like gene activation with high dynamic ranges using σ54- dependent promoters.
A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules
Bacterial allosteric transcription factors can sense and respond to a variety of small molecules. Here the authors present CaT-SMelor which uses Cas12a and allosteric transcription factors to detect small molecules in the nanomolar range.
Single-particle tracking PALM (sptPALM) provides quantitative information in vivo if the protein of interest remains in a single diffusional state during track acquisition. Here the authors develop a custom-built sptPALM microscope and a Monte-Carlo based diffusion distribution analysis to study dynamic DNA-dCas9 interactions in live bacteria.
Cytosine base editors are limited by editing scope and potential off-target effects. Here the authors screen diversified lamprey cytidine deaminases along with different protein fusion architectures and present base editors with improved fidelity.
Cystic fibrosis is caused by mutations in the CFTR chloride channel. Here, the authors develop a gene therapy approach using the programmable nuclease AsCas12a to correct a splicing mutation in CFTR, and show efficient repair of the mutation and recovery of CFTR function in patient-derived organoids and airway epithelial cells.
Conventional methods to detect AAV vector transduction can miss transient or low levels of reporter expression. Here the authors use editing-reporter mice and discover numerous sites of AAV targeting along with better prediction of the gene editing footprint.
Manipulating DNA repair pathways can be used to improve the outcomes of CRISPR-based genome editing. Here the authors derive an enhanced RAD18 variant that suppresses 53BP1 recruitment to DNA double-strand breaks to enhance homology-mediated repair.
AsCpf1 is an alternative nuclease to Cas9 for CRISPR mediated genome engineering. Here the authors demonstrate functional genomic screens with AsCpf1 that minimize library size with no loss in gene targeting efficiency.
Here, the authors show that sequential treatment with long-acting slow-effective release ART and AAV9- based delivery of CRISPR-Cas9 results in undetectable levels of virus and integrated DNA in a subset of humanized HIV-1 infected mice. This proof-of-concept study suggests that HIV-1 elimination is possible.
When Lama1 was upregulated using CRISPR and a catalytically inactive Cas9 in a mouse model of congenital muscular dystrophy type 1A, apparent hindlimb paralysis, muscle fibrosis and nerve myelination defects were ameliorated in symptomatic mice.
Cytosine and adenine base editors have undesired off-target effects on RNA, but this activity can be reduced in deaminase-engineered variants while preserving on-target DNA editing.
AAV-mediated CRISPR–Cas9 therapy extends lifespan and ameliorates disease-related phenotypes in a mouse model of Hutchinson–Gilford progeria syndrome.
A programmable transposase integrates donor DNA at user-defined genomic target sites with high fidelity, revealing a new approach for genetic engineering that obviates the need for DNA double-strand breaks and homologous recombination.
AAV-mediated CRISPR/Cas9 therapy extends life span and ameliorates disease-related phenotypes in a mouse model of Hutchinson–Gilford progeria syndrome.
Optimized conditions for ribonucleoprotein delivery of Cas9–sgRNA complexes enables precise and efficient gene editing to restore fetal hemoglobin expression in sickle cell disease patient-derived HSCs
Screening of Cas9 and gRNA combinations identifies the variant SaCas9-KKH that selectively disrupts the mutant Tmc1/TMC1 allele that causes hearing loss, prevents deafness and preserves inner-ear hair cell function in Tmc1 mutant Beethoven mice.
In the mdx mouse model of Duchenne muscular dystrophy, single intravenous administration of AAV-CRISPR–Cas9 vectors provides efficient genome editing and restoration of dystrophin expression lasting for one year.
Few approaches for targeted manipulation of the epigenome are available in plants. Here, the authors adapt the dCas9-SunTag system to engineer targeted gene activation and site-specific manipulation of DNA methylation in Arabidopsis.
The development of mobile CRISPR interference (CRISPRi), a modular dCas9-based system that facilitates blocking of gene expression and is easily transferred via conjugation, enables genetic investigations in non-model bacteria.
One year after the world learned of He Jiankui’s editing of twins, gaps in rules remain.
CRISPR–Cas systems have revolutionized genome editing, and the CRISPR–Cas toolkit has been expanding to include single-base editing enzymes, targeting RNA and fusing inactive Cas proteins to effectors that regulate various nuclear processes. Consequently, CRISPR–Cas systems are being tested for gene and cell therapies.
Companies are marrying gene editing with stem cell therapies in new clinical trials.
Genome editing through direct editing of bases holds promise for achieving precise genomic changes at single-nucleotide resolution while minimizing the occurrence of potentially mutagenic double-strand DNA breaks. In this Review, Rees and Liu provide a comprehensive account of the state of the art of base editing of DNA and RNA, including the progressive improvements to methodologies, understanding and avoiding unintended edits, cellular and organismal delivery of editing reagents and diverse applications in research and therapeutic settings.
News and views
A new genome editing strategy called prime editing uses a catalytically impaired Cas9 fused to an engineered reverse transcriptase to write desired genetic sequence information directly into a target locus.
Bacteria that infect rice are thwarted by changes to rice genes involved in sugar transport.
The system allows researchers more control over DNA changes, potentially opening up conditions that have challenged gene-editors.
Russian ‘CRISPR-baby’ scientist has started editing genes in human eggs with goal of altering deaf gene
Denis Rebrikov also told Nature that he does not plan to implant gene-edited embryos until he gets regulatory approval.
Researchers rapidly corrected finding through discussions on social media and preprints.
Researchers are using the gene-editing tool to boost the fruit’s defences and prevent the extinction of a major commercial variety.
Two recent studies report innovative RNA base editing tools: adenine editing without the requirement for exogenous enzymes, and a new ability to edit cytosines.
Scientists struggle to detect the unauthorized sale of gene-edited crops whose altered DNA can mimic natural mutations.
Parasitic genetic elements called transposons carry CRISPR machinery that is normally used against them by bacterial cells. This paradox has now been explained, with implications for gene-therapy research.
Efforts to make heritable changes to the human genome are fraught with uncertainty. Here’s what it would take to make the technique safe and acceptable.
Another researcher has announced controversial plans to gene edit babies. The scientific community must intervene.
The proposal follows a Chinese scientist who claimed to have created twins from edited embryos last year.
Study of almost half a million people links mutation that protects against HIV infection to an earlier death.