Genome engineering

Optimized base editors targeting the exon-7 mutation in SMN2 restore expression of the survival motor neuron (SMN) protein to normal levels, as shown in mice with spinal muscular atrophy and in fibroblasts from patients with this genetic disease.

A method leveraging an integrase-deficient lentivirus, homology-directed repair and the electroporation of a CRISPR-associated ribonucleoprotein complex allows for the knock-in and stable expression of large payloads in primary human cells.

The yields of edited primary human lymphocytes can be increased substantially, with respect to those obtained via electroporation, by delivering a CRISPR ribonucleoprotein alongside an amphiphilic peptide identified via screening.

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 lentiviral vector incorporating two functionally independent promoters allows for the generation of T cells that express a tumour-directed receptor constitutively and an inducible gene that is expressed only in the presence of a target antigen.

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.

A library of double-stranded-DNA deaminase-derived cytosine base editors allows for the precise ablation of every mtDNA protein-coding gene in the mouse mitochondrial genome.

Göttingen minipigs genetically engineered to carry a mini-repertoire of human genes for immunoglobulin antibodies allow for the toxicological testing of human recombinant antibodies.

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.

In mouse models of hereditary tyrosinaemia and of Leber congenital amaurosis, prime editing can precisely correct the disease-causing mutations, ameliorating the disease phenotypes.

Aminoacyl-tRNA-synthase–tRNA pairs specific for the desired unnatural amino acids can read through a nonsense mutation in the dystrophin gene, and partially restore muscle function in mice.

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.

Cytidine base editors delivered to the liver of mice with phenylketonuria via adeno-associated viruses or lipid nanoparticles do not lead to detectable off-target edits in the RNA and DNA of hepatocytes.

Lentiviruses co-packaging SpCas9 mRNA and an expression cassette encoding for a guide RNA targeting the Vegfa gene prevent mice from developing wet age-related macular degeneration induced by Vegfa. [Summary amended to correct the description of the packaged cargo.]

Subretinal viral delivery, in mice, of an adenine base editor and a single-guide RNA targeting a nonsense mutation in the Rpe65 gene restores near-normal levels of retinal and visual functions.

A web tool enables the rapid design of prime-editing guide RNAs from reference and edited DNA sequences, for diverse prime-editing applications.

Pigs can be genetically modified to inactivate endogenous retroviruses and to display enhanced compatibility with the human immune system using a combination of CRISPR–Cas9 and transposon technologies.

A comparison of compatibilities in protospacer adjacent motifs and of on-target and off-target activities of Streptococcus pyogenes Cas9 variants at endogenous sites in human cells enables the editing of new genomic sites associated with genetic diseases.

Optimized adeno-associated viruses delivering split cytosine base editors and adenine base editors with trans-splicing inteins can edit brain, liver, retina, heart and skeletal-muscle tissues at therapeutically relevant efficiencies.

Orthologous therapeutic proteins can elude the adaptive immune system and allow for effective repeated dosing, as shown for CRISPR gene editing with orthologues of adeno-associated viral capsids and of the Cas9 protein.

Intravenous delivery of an adenine base editor and a single-guide RNA for the Fah gene can correct an A>G splice-site mutation in an adult mouse model of tyrosinaemia.

Genome-edited human pluripotent stem cells and genome-edited mouse models reveal that combinatorial genetic interactions contribute to the complex genetic heritability of human cardiomyopathy.

CRISPR–Cas9-mediated genome editing can be activated locally in vivo via an applied magnetic field, after complexation of magnetic nanoparticles with recombinant baculoviral vectors packaging the CRISPR–Cas9 machinery.

The transplantation of skin cells genetically modified to express an enzyme that hydrolyses cocaine into mice leads to long-lasting levels of the enzyme in the circulation and protects the mice from cocaine-seeking behaviour and cocaine overdose.

Gene editing of a single gene in the brain of an adult mouse model of fragile X syndrome, achieved via the intracranial injection of a nonviral Cas9 delivery vehicle, rescues mice from the exaggerated repetitive behaviours caused by the disease.

A cloud-based machine-learning software that scores individual guide–target pairs and provides an overall summary score for a given guide that outperforms competing algorithms for the prediction of CRISPR–Cas9 off-target effects.

A model accounting for the properties of the local chromatin environment predicts the modulation of patterns in gene expression and helps screen for chemotherapeutic adjuvants that lead to an enhanced therapeutic response in cancer cells.

The expression of two specific DNA-repair factors promotes homology-directed repair and enhances the precision of CRISPR–Cas9 gene editing at multiple loci in human cells, including patient-derived induced pluripotent stem cells.

Gold nanoparticles carrying Cas9 ribonucleoprotein and donor DNA, and complexed with endosomal disruptive polymers, correct the DNA mutation that causes Duchenne muscular dystrophy in mice, with minimal off-target effects.

An array of chemically engineered CRISPR RNAs and AsCpf1 messenger RNAs leads to improvements in gene-cutting efficiency up to about 300% with respect to unmodified CRISPR RNA and plasmid-encoding AsCpf1.

The continuing optimization of the safety and applicability of CRISPR for ex vivo genome editing will broaden the prospects of cell therapies.

Amphiphilic peptides can aid the delivery of CRISPR ribonucleoproteins into primary human lymphocytes at low toxicity, boosting editing yields with respect to the use of electroporation.

Prime editing can efficiently rewrite the genetic mutation causing sickle cell disease, in haematopoietic stem cells from patients.

Each of the 13 protein-coding genes in the mouse mitochondrial genome can be ablated using a library of optimized double-stranded-DNA deaminase-derived cytosine base editors.

The preclinical performance of subretinal or intracorneal delivery of Cas9 nucleases encoded in RNA foreshadows safer and effective one-and-done gene therapies for eye diseases.

Subretinal delivery of lentiviruses bearing Cas9 mRNA and a guide RNA targeting the Vegfa gene reduces the development of choroidal neovascularization in a mouse model of wet age-related macular degeneration.

The sustained expression of RNA-targeting Cas9 delivered intramuscularly or systemically by adeno-associated viral vectors eliminates pathogenic foci of expanded-repeat transcripts and reverses muscle-disease phenotypes in mouse models of myotonic dystrophy type 1.

By using CRISPR and transposon constructs, pigs have been genetically modified to inactivate endogenous retroviruses and to enhance the compatibility of their organs with the human immune and coagulation systems.

In less than a decade, the genome-editing technology now recognized by the Nobel Prize in Chemistry has impacted the biological and biomedical sciences widely. What’s next for CRISPR in biomedicine?

The editing of single DNA bases in the genome is being optimized for higher editing precision and versatility.

In an adult mouse model of tyrosinaemia, a base editor correcting an A-to-G splice-site mutation in the Fah gene restores the translation of the functional enzyme, promoting the repopulation of the liver with the corrected cells.

Orthologues of CRISPR-associated proteins and of viral vectors evade immune recognition in mice, enabling repeated gene therapy.

The simplicity and powerful capabilities of CRISPR have led to an explosion of genome-editing applications. Their continued development should be nurtured by scientific and legal environments that discourage and penalize irresponsible uses of the technology.

Graftable skin expressing an enzyme that metabolizes cocaine may offer a new strategy to treat cocaine addiction.

Magnetic nanoparticles complexed with recombinant baculoviral vectors containing the CRISPR–Cas9 machinery enable the local magnetic activation of genome editing.

Human cardiomyocytes and mice edited to harbour gene variants found in patients with dilated cardiomyopathy reveal combinatorial genetic interactions that contribute to the complex genetic heritability of the disease.

Widespread editing of the mutated DMD gene by CRISPR–Cas9, systemically delivered via an adeno-associated virus, restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

The optimization and diversification of methods for manipulating the genome will enable new therapeutic solutions.

A theoretical model of chromatin packing heterogeneity predicts patterns in gene expression and can be used to screen for effective chemotherapeutic adjuvants.

The expression of two DNA repair factors improves the recombination of single-stranded oligodeoxynucleotides with Cas9-induced double-strand breaks, facilitating precise and efficient gene editing.

The DNA mutation that causes Duchenne muscular dystrophy in mice can be corrected, with minimal off-target effects, by gold nanoparticles carrying the CRISPR components.