Casgevy (exagamglogene autotemcel) was the first CRISPR-based gene therapy to receive regulatory approval, in 2023, for the treatment of sickle cell disease and of transfusion-dependent β-thalassemia1. Other ex vivo CRISPR-based therapies, for multiple myeloma and other cancers, as well as in vivo gene editing in the eye (to target common pathogenic variants), are progressing through early-stage clinical development2. And, so far, human trials of base editing have shown encouraging outcomes: trials for sickle cell disease (BEAM-101, using base-edited autologous haematopoietic stem cells to induce fetal haemoglobin), for relapsed or refractory T cell acute lymphoblastic leukaemia and lymphoma (BEAM-201, using multiplexed base editing of allogeneic chimeric antigen receptor (CAR) T cells) and for heterozygous familial hypercholesterolemia (VERVE-101, involving a lipid-nanoparticle delivery system for the in vivo inactivation of the PCSK9 gene in the liver) have reported generally positive preliminary data. In 2022, the compassionate use of base-edited CAR T cells successfully treated relapsed T cell acute lymphoblastic leukaemia in a 13-year-old girl and resulted in remission3.

Adapted from the Article by Sousa et al., under a Creative Commons license CC BY 4.0.

Initially celebrated for their ability to induce targeted double-strand DNA breaks, CRISPR-based methods have evolved, rather rapidly, to encompass a far broader repertoire of functionalities, including precise base substitutions, targeted insertions and deletions, and the modulation of gene expression. Fuelled by a deeper understanding of DNA-repair pathways and by a growing toolkit of engineered Cas proteins and guide RNAs, genome editors are increasingly enabling therapeutic applications with higher degrees of precision and control. Notably, base editing and prime editing can be used to directly modify DNA sequences without inducing double-strand DNA breaks, which reduces the risk of some unintended editing outcomes (such as uncontrolled indels or large deletions) and increases the fidelity of targeted gene correction. By leveraging DNA-repair and DNA-replication machineries, base editing and prime editing can also lead to high editing efficiencies in non-dividing cells. This is a crucial advantage for therapeutic applications targeting neurons, cardiac or skeletal muscle cells and other post-mitotic cell types.

Advances in the editing machinery are being coupled with innovations in delivery. Viral vectors, although effective for some applications (particularly when the efficient targeting of specific cells is paramount), come with constraints in packaging capacity and immunogenicity. Lipid nanoparticles, cell-penetrating peptides and ribonucleoprotein complexes are being intensively explored, as they offer advantages at least on three fronts: transient and controlled expression of the genome editors (thus reducing the opportunity for off-target edits), minimization of any off-target effects (because of the transient activity) and enhanced safety (by avoiding permanent genomic alterations arising from viral integration). The convergence of enhanced editing capabilities and sophisticated delivery methods is driving the development of genome-editing therapies for the treatment of a wider range of genetic diseases, from metabolic disorders and hereditary deafness to cystic fibrosis and inherited retinal degeneration.

This issue of Nature Biomedical Engineering highlights advances that may power genome-editing technologies of human trials in a not-to-distant future. The advances aim to enhance the efficiency and precision of base editing and prime editing, to overcome delivery challenges, to expand the scope of genome-editing applications, to address safety concerns and to take advantage of DNA-repair mechanisms.

Specifically, Sangsu Bae and colleagues investigated the prevalence of large deletions, arising at Cas9-induced double-strand breaks as well as at single-strand breaks produced by Cas9 nickases, across diverse cell types (and, importantly for therapeutic applications, in human primary T cells). They show that base editors and prime editors generated large deletions at approximately 20-fold lower frequency than Cas9 nucleases and that small-molecule inhibitors of the involved DNA-repair pathways can reduce the frequency of these undesirable events. Erwei Zuo and colleagues describe how structure-guided protein engineering (with the aid of Alphafold2, ref. 4) can be used to identify cytidine deaminases with higher editing efficiencies, broader editing windows and reduced off-target effects. Such deaminases with reduced sequence-context independence coupled with rational mutagenesis may help to further minimize off-target editing, may aid targeted gene modulation and are likely to enhance the specificity of base-editor therapies.

Two studies by David Liu and collaborators also included in this issue provide advantageous techniques for the delivery of large DNA sequences into cells, and for improving the delivery efficiency and potency of base editors and prime editors. In one article, the researchers report that phage-assisted continuous evolution can be leveraged to engineer enhanced Bxb1 recombinases that substantially improve the efficiency of the site-specific integration of large DNA payloads (exceeding 10 kilobases) in mammalian cells. The method can be used for enhancing targeted gene insertion, replacement or correction, with respect to what can be achieved via homology-directed repair. In a second article, Krzysztof Palczewski, David Liu and co-authors report optimized lipid nanoparticle formulations (by screening a panel of ionizable cationic lipids and optimizing the concentration of a synthetic lipid) for the delivery of ribonucleoproteins for base editing and prime editing, where the improvements in in vivo editing efficiency led to enhanced ribonucleoprotein stability and delivery efficiency, as well as increased editing potency and reduced off-target effects in vivo. Formulations for the delivery of proteinic genome-editing effectors will incentivize the further development of transient and thereby likely safer gene therapies.

Two other studies report the feasibility of base editing and prime editing for two particularly challenging therapeutic applications: the long-term restoration of auditory function in mice with a recessive form of profound deafness (Yilai Shu and co-authors used an adenine base editor, delivered by an adeno-associated virus, to target a prevalent mutation in the Otof gene) and the efficient functional correction of the predominant cause of cystic fibrosis (the CFTR F508del mutation) in primary airway epithelial cells from patients (Liu and co-authors took advantage of systematic efficiency optimizations for prime editing, from engineered prime-editing guide RNAs and variants of prime editors to strategic silent edits to the transient expression of a dominant-negative mismatch-repair protein).

The issue also includes two high-throughput screening methods that allow for a more efficient exploration of gene function and therapeutic targets. Adriano Aguzzi and co-authors describe arrayed CRISPR libraries (generated using automated liquid-phase assembly) for the genome-wide activation, deletion and silencing of human protein-coding genes. The libraries allow for the systematic perturbation of gene function and for the identification of disease modifiers, as they show for previously unknown regulators of a cellular prion protein. Prashant Mali and colleagues used combinatorial screening for the identification and engineering of deimmunized Cas9 variants. Specifically, they created a Cas9 variant with seven simultaneously silenced immunogenic epitopes, which would facilitate the safety of therapeutic applications where persistence of the nuclease is necessary or unavoidable.

In aggregate, the eight studies highlighted here paint a meaningful picture of the ongoing evolution of genome-editing technologies: improvements in the precision and efficiency of genome-editing techniques, an increased expansion of genome-editing applications (from correcting point mutations to inserting large DNA sequences to modulating gene expression) and the development of more powerful screening methods and in vivo delivery systems. And with enhanced precision and potency, promise may translate into progress.