SCGE

This Perspective discusses how the Somatic Cell Genome Editing Consortium aims to accelerate the implementation of safe and effective genome-editing therapies in the clinic.

Gene therapy is at an inflection point. Recent successes in genetic medicine have paved the path for a broader second wave of therapies and laid the foundation for next-generation technologies. This comment summarizes recent advances and expectations for the near future.

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.

The scientific, technical and ethical aspects of using CRISPR technology for therapeutic applications in humans are discussed, highlighting both opportunities and challenges of this technology to treat, cure and prevent genetic disease.

A growing arsenal of CRISPR-based tools enables increasingly sophisticated genome editing applications.

As CRISPR therapies move into clinical testing, David Schaffer and colleagues review a raft of different delivery technologies being road tested to address cargo capacity limitations, maximize potency, minimize off-target effects and avoid immunogenicity.

A custom adenine base editor can edit the variant of the β-globin gene that causes sickle cell disease into a non-pathogenic variant in human and mouse cells, and transplantation of the edited cells rescues sickle cell disease in mice.

The genome-wide activity of Cas9 is measured in unprecedented detail.

Orange CaMBI, a genetically encoded bioluminescent calcium indicator consisting of calcium-sensing domain CaM, luciferase, and fluorescent proteins, reports calcium dynamics in single cells and reveals calcium oscillations in whole mouse organs.

An integrative three-dimensional genomic and transcriptional profiling of four human neural cell types links regulatory elements to their target genes and elucidates the function of noncoding variants in neuropsychiatric disorders.

This protocol describes CIRCLE-seq (circularization for in vitro reporting of cleavage effects by sequencing), a sensitive and unbiased method for defining the on-target and off-target activity of CRISPR–Cas9 nucleases genome-wide.

Off-target effects of programmable nucleases remain a critical issue for therapeutic applications of genome editing. This review compares experimental and computational tools for off-target analysis and provides recommendations for better assessments of off-target effects.

In a mouse model of progeria, an adenine base editor delivered with adeno-associated virus corrects the pathogenic mutation in LMNA, rescues vascular pathology and markedly extends the lifespan of the mice.

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.

Understanding the risks of bystander and off-target editing is essential for genome engineering applications. Here, the authors analyze the fidelity of adenine and cytosine base editors in vivo.

Two conditional transgenic mouse lines based on CRISPRa and CRISPRi enable epigenome editing in vivo.

The Cas9-specific T cell response has been speculated to impair CRISPR therapy. Here, the authors show that local and systemic AAV CRISPR therapy induces cytotoxic killing and eliminates rescued dystrophin in canine models of Duchenne muscular dystrophy.

Wang et al. describe the development of a 3D in vitro model of human skeletal muscle that recapitulates the pathophysiology of Pompe disease. They further define a Pompe disease-specific transcriptional signature, confirm its presence in the Pompe myobundles, and detect a partial reversal of the signature upon in vitro treatment of myobundles with rhGAA.

Differentiation of hPSCs to cardiomyocytes suffers from high variability. Here the authors report a label-free live cell imaging platform based on autofluorescence imaging to enable the prediction of cardiomyocyte differentiation efficiency from hPSCs.

Mutations in CNTNAP2 have been associated with a syndromic form of Autism Spectrum Disorder. Here the authors show that forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of ASD with a homozygous truncating mutation in CNTNAP2 displayed an increase in volume and total cell number, which is driven by abnormal cellular proliferation and neurogenesis.

Mutations in the splicing factor RBM20 cause aggressive Dilated Cardiomyopathy. Here the authors generated RBM20 R636S mutants and knockout in human iPSC-derived cardiomyocytes. Mutant RBM20 showed different target RNA binding, altered splicing and localization to cytoplasmic processing bodies.

Samelson et al. report that BRD2 inhibition suppresses SARS-CoV-2 infection in epithelial cells and Syrian hamsters via reducing the transcription of host cell surface receptor ACE2.

In vivo assessment of nuclease off-target activity has primarily been indirect or through ChIP-based detection of double-strand break DNA repair factors, which can be cumbersome. Here, the authors show that GUIDE-tag, enables one-step off-target genome editing analysis in mouse liver and lung.

Tissues and organoids derived from human pluripotent stem cells with knocked-out kinesin-2 subunits lack cilia, and can be used to model ciliopathy phenotypes and to reveal underlying mechanisms of disease.

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.

Genomic analyses of major clades of huge phages sampled from across Earth’s ecosystems show that they have diverse genetic inventories, including a variety of CRISPR–Cas systems and translation-relevant genes.

An interbacterial toxin that catalyses the deamination of cytidines within double-stranded DNA forms part of a CRISPR-free, RNA-free base editing system that enables manipulation of human mitochondrial DNA.

Wider editing windows and different PAM requirements enable a broad set of genomic positions to be targeted with A and C base editors.

Improved base editors are generated by continuous evolution.

PAM sequences without G bases can be edited with SpCas9 variants that were continuously evolved in the laboratory.

Methods to efficiently detect Cas9-independent cytosine base editor off-target activity enable the identification and development of variants with minimal off-target editing and robust on-target editing.

Adeno-associated viruses (AAVs) can be targeted in a tissue-specific manner, but their tissue accumulation cannot be assessed in a non-invasive manner. Here the authors conjugate a multivalent chelator labelled with Cu-64 to the surface of AAVs and image the brain accumulation of the PHB.eB capsid by PET.

The most abundant RNA modification in humans, m⁶A, can be installed at specified RNA sequences in cells, enabling functional studies.

Efficiency of transversion base editing is increased by matching a set of base editors to target sequences.

Moghadam et al. developed a CRISPR transcriptional repressor to silence MyD88 expression in vivo to modulate immune response against AAV gene therapy and septicaemia.

A continuously evolved adenine base editor is compatible with various Cas proteins and mediates efficient A•T-to-G•C base conversions at a wide variety of PAM sites.

Prime editing guide RNA design is more complex than for standard CRISPR-based nucleases or base editors. Here the authors present PrimeDesign and PrimeVar for the rapid and simplified design of pegRNA and ngRNA combinations.

Prime editors use a template sequence within their pegRNA to facilitate nucleotide substitutions or local indels. Here the authors use AAVs to deliver a split-intein prime editor in vivo to correct a pathogenic mutation.

This phage-assisted continuous evolution protocol enables directed protein evolution. It includes preparation of selection phage and host cells, assembly of a continuous flow apparatus and performance and analysis of evolution experiments.

In a mouse model of progeria, an adenine base editor delivered with adeno-associated virus corrects the pathogenic mutation in LMNA, rescues vascular pathology and markedly extends the lifespan of the mice.

Cryo-EM structures of CRISPR–CasΦ, a small RNA-guided enzyme unique to bacteriophages, reveal how CasΦ binds to and cleaves DNA, paving the way for engineering of improved CasΦ variants for diagnostics and genome-editing applications.

Here the authors fuse hRad51 and variants thereof to Cas9 nickase to facilitate homology-directed repair without generating double strand breaks, minimizing indel formation and off-target editing. This tool represents progress towards the goal of performing HDR without an excess of undesired side products.

Prime editors are optimized for use in plants.

This historical Perspective on continuous directed evolution focuses on laboratory approaches that enable greater understanding of evolving molecular populations and offer investigators tools to guide the emergence of new biomolecular systems.

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.

Stabilizing pegRNAs with 3′ RNA structures increases prime editing efficiency.

The directed evolution of antibodies yields important tools for research and therapy. Here the authors develop a periplasmic phage-assisted continuous evolution platform for improvement of protein-protein interactions in the disulfidecompatible E. coli periplasm.

Prime editing of large DNA sequences is achieved with two pegRNAs and site-specific recombinases.

Continuously evolved double-stranded DNA deaminases increase the efficiency and targeting scope of all-protein base editors.

Leber congenital amaurosis is caused by mutations in RPE65 and leads to retinal degeneration in children. Here, the authors show that in vivo base editing can prolong the survival of cone photoreceptors and rescue their function in a mouse model of the disease.

Here, the authors use cryo-EM and biochemical analysis to investigate how the CRISPR-associated protein Cas9 interrogates DNA to locate its RNA-matching target sequence.

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.

Diseases caused by different mutations in the two alleles of a gene are treated in mice by Cas-9-induced allelic exchange.

The most abundant RNA modification in humans, m⁶A, can be installed at specified RNA sequences in cells, enabling functional studies.

Inefficient delivery of proteins into mammalian cells limits their use in research and therapy. Here, the authors discover that ProTα, a small, intrinsically disordered human protein can facilitate efficient cationic lipid-mediated protein delivery of genome editing proteins into mammalian cells.

The adaptor protein SHN3 suppresses new bone formation by controlling osteoblast activity. Here, the authors show that ablation of SHN3 function, either genetically or by delivering an artificial miRNA via AAV9, rescues bone loss in osteoporotic mice, and show that engineering of the AAV9 capsid improves targeting to bone

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.

Adeno-associated viruses (AAVs) can be targeted in a tissue-specific manner, but their tissue accumulation cannot be assessed in a non-invasive manner. Here the authors conjugate a multivalent chelator labelled with Cu-64 to the surface of AAVs and image the brain accumulation of the PHB.eB capsid by PET.

Long-term expression of Cas9 following precision genome editing in vivo may lead to undesirable consequences. Here we show that a single-vector, self-inactivating AAV system containing Cas9 nuclease, guide, and DNA donor can use homology-directed repair to correct disease mutations in vivo.

Polymer nanocapsules, assembled around a ribonucleoprotein complex of Cas9 nuclease and a single-guide RNA, enable safe and efficient gene editing in vitro and in vivo.

Adeno-associated virus (AAV) vectors represent the leading platform for therapeutic gene delivery, with two recombinant AAV gene therapy products having gained regulatory approval in Europe or the United States. Here, Gao and colleagues discuss the fundamentals of AAV and vectorology, focusing on current therapeutic strategies, clinical progress and ongoing challenges.

Cas9 base editors are promising tools for correcting pathogenic single nucleotide mutations. Here the authors chemically modify mRNA encoding the editor and the gRNA to enhance editing and broaden its application.

Adeno-associated viruses (AAVs) are vehicles for gene therapy in humans, but currently only a limited amount of AAV serotypes is available. Here, the authors identify a novel AAV, AAVv66, and demonstrate enhanced production yields, virion stability, and CNS transduction compared to the clinically approved serotype AAV2.

Lipid nanoparticles can be optimized for the efficient delivery of therapeutic mRNAs to the lung via nebulization, as shown for the delivery of a therapeutic antibody in mice challenged with a lethal dose of the H1N1 influenza A virus.

Prime editing is expanded to deletions and replacements of genomic sequences of up to 10 kb.

Worldwide pandemics of obesity and diabetes prompt an urgent need for new approaches to their prevention and cure. Here the authors present a CRISPR-based strategy that enhances the therapeutic potential of human adipocytes when implanted in obese mice.

The authors developed AAV capsids for robust transgene expression in the brain with decreased liver targeting after non-invasive administration in mice and marmosets, enabling more targeted systemic gene delivery to the brain.

Inflammation plays a central role in our body’s response to injury or infection. If dysregulated, inflammatory responses can lead to chronic inflammation and the development of inflammatory diseases. This Review discusses biomaterials-based anti-inflammatory therapies, including scavenging, blockage and drug delivery strategies.

The extent to which mRNA delivery, as well as the cellular response to mRNA drug delivery vehicles, is conserved across species in vivo is unknown. Using species-independent DNA barcoding, the authors measure delivery in humanized, primatized and normal mice, and identify a potential mechanism driving species-dependent lipid nanoparticle delivery.

The feasibility of adeno-associated-virus-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) is explored to restore gene function, using a mouse model of mucopolysaccharidosis type I for proof of concept.

A split prime editor simplifies delivery.

Despite an emerging role for cerebrovascular endothelial cells in a range of neurological pathologies, AAV vector development to date has focused on tools designed to target neurons or astrocytes. Here, Krolak et al. describe a specific variant of AAV (AAV-BI30), with high specificity and efficacy for transduction of endothelial cells across the central nervous system.

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.