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.
Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency
Precise genome editing is made more efficient by stabilizing Cas9 and enhancing shuttling to the nucleus.
A type I CRISPR–Cas system, rather than the conventional type II CRISPR systems, is adapted for gene editing by fusing Cascade to the FokI nuclease domain.
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.
Genome editing of three SWEET gene promoters endows rice with resistance to all Xanthomonas bacterial blight strains tested.
One third of verified gene knock outs with CRISPR still show residual protein expression owing to translation reinitiation or exon skipping. Several proteins are still functional. The authors call for a systematic analysis of protein levels after genome editing.
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.
The Alu editing index (AEI) quantifies genome-wide editing in Alu repeats and allows comparison across samples.
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.
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.
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 activation (CRISPRa) can target select genes and, rather than being used to delete them, can be used to activate their expression. Chen and colleagues use a CRISPRa-based approach to drive the expression of multiple endogenous genes in tumors and presentation of the antigens encoded, thus enhancing antitumor immune responses.
A genome-edited hornless compound heterozygous dairy bull transmitted this trait to his offspring.
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.
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.
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.
Immunomagnetic cell sorting implemented in a microfluidic chip can perform loss-of-function CRISPR–Cas9-mediated phenotypic screening at higher throughput than fluorescence-activated cell sorting.
Type I CRISPR–Cas systems, the largest group of CRISPR systems in nature, can be repurposed for DNA targeting and gene regulation in human cells
In vivo CRISPR screening in CD8 T cells with AAV–Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma
A hybrid AAV–transposon CRISPR approach enables large-scale in vivo screens in T cells and identifies potential targets for immunotherapy.
In addition to causing A-to-G base transitions, adenine base editors also cause C base substitutions
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.
Hidden antibiotics in actinomycetes can be identified by inactivation of gene clusters for common antibiotics
Dereplication by inactivation of antibiotic production clusters in actinomycetes enables antibiotic discovery.
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.
Engineered variants of DNA base editors have reduced RNA off-target editing while retaining DNA on-target efficiency.
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.
A single transcript encoding Cas12a and an array of CRISPR RNAs enables multiplexed genome engineering, from multiple knockouts to transcriptional activation or repression to orthogonal transcriptional control and editing in the same sample.
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.
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.
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.
A machine learning model based on data from primary human T cells accurately predicts repair outcomes after CRISPR–Cas9 editing.
Engineered zinc finger nucleases achieve 98% knockout of a target gene in human T cells without detectable off-target activity.
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.
Improved base editors are generated by continuous evolution.
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.
CombiSEAL is a high-throughput platform for seamlessly assembling barcoded combinatorial genetic units, offering an approach for protein optimization such as screening SpCas9 variants.
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.
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.
A Review informatively summaries the recent development and breakthroughs of CRISPR technology, with a focus on progresses, challenges and potential utility in plant science.
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.
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
Excitement builds around press-released data from two individuals treated with a CRISPR–Cas9-based therapy targeting somatic cells.
The simultaneous removal of endogenous T-cell receptor α-chains and β-chains, and the orthotopic placement of an exogenous receptor in human T cells via CRISPR gene-editing, prevents the mispairing between endogenous and transgenic receptors while preserving the cells’ function.
Early results suggest that prime editors are cleaner than CRISPR–Cas9 and more versatile than base editors, but many questions remain.
Bacteria that infect rice are thwarted by changes to rice genes involved in sugar transport.
Two studies offer a powerful strategy for combating bacterial blight in rice.
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.
One year after the world learned of He Jiankui’s editing of twins, gaps in rules remain.
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.
Haematopoietic stem and progenitor cells, edited via CRISPR to mimic mutations ameliorating sickle-cell disease, engraft when transplanted in macaques and maintain foetal haemoglobin in circulation.
Researchers rapidly corrected finding through discussions on social media and preprints.
Orthologues of CRISPR-associated proteins and of viral vectors evade immune recognition in mice, enabling repeated gene therapy.
Small players take on big seed conglomerates with next-generation non-GMO crops.
Researchers are using the gene-editing tool to boost the fruit’s defences and prevent the extinction of a major commercial variety.
Bluebird Bio’s gene therapy eliminates the need for blood transfusions in patients with β-thalassemia.
Two recent studies report innovative RNA base editing tools: adenine editing without the requirement for exogenous enzymes, and a new ability to edit cytosines.
CRISPR–Cas-associated transposases enable targeted integration of new sequences into genomes.
Scientists struggle to detect the unauthorized sale of gene-edited crops whose altered DNA can mimic natural mutations.