Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene-editing system (CRISPR-Cas) is a valuable tool for fundamental and applied research applications. Significant improvements in editing efficacy have advanced genome editing strategies into phase 3 human clinical trials. However, recent studies suggest that our understanding of editing outcomes has lagged behind the developments made in generating the edits themselves. While many researchers have analyzed on- and off-target events through the lens of small insertions or deletions at predicted sites, screens for larger structural variants (SVs) and chromosomal abnormalities are not routinely performed. Full and comprehensive validation of on- and off-target effects is required to ensure reproducibility and to accurately assess the safety of future editing applications. Here we review SVs associated with CRISPR-editing in cells of human origin and highlight the methods used to detect and avoid them.

Skin wound healing is a crucial process for regenerating healthy skin and avoiding the undesired consequences associated with open skin wounds. For epidermolysis bullosa (EB), a debilitating group of fragile skin disorders currently without a cure, skin blistering can often be severe and heal poorly, increasing susceptibility to life-threatening complications. To prevent these, investigational therapies have been exploring the use of tissue-engineered skin substitutes (TESSs) aimed at replacing damaged skin and promoting long-term wound closure. These products have either been developed in house or commercially sourced and are composed of allogeneic or autologous human skin cells, often with some form of bioscaffolding. They can be broadly classified based on their cellular composition: keratinocytes (epidermal substitutes), fibroblasts (dermal substitutes) or a combination of both (composite substitutes). Encouraging long-term wound healing has been achieved with epidermal substitutes. However, these substitutes have not demonstrated the same efficacy for all patients, which may be due to the molecular heterogeneity observed between EB subtypes. Autologous composite TESSs, which more closely resemble native human skin, are therefore being investigated and may hold promise for treating an extended range of patients. Additionally, future TESSs for EB are focused on using gene-corrected patient skin cells, which have already demonstrated remarkable long-term wound healing capabilities. In this review, we provide an overview of the different TESSs that have been investigated in clinical studies to treat patients with EB, as well as their long-term wound healing results. Where available, we describe the methods used to develop these products to inform future efforts in this field.