A research team led by Luigi Nardini at the San Raffaele Telethon Gene Therapy Institute (SR-Tiget) has developed a new strategy that significantly improves the accuracy and safety of CRISPR-Cas9 gene editing in human blood stem cells, potentially overcoming one of the major barriers limiting widespread clinical application of genome editing therapies.
This research nature biotechnologypresent SMArT (“artificial transactivator-mediated selection”), an innovative platform to achieve targeted integration of gene-sized cassettes and validate the results of the procedure. Using this strategy, edited hematopoietic stem and progenitor cells (HSPCs) can be enriched to near purity while selectively removing cells containing unintended and potentially deleterious genomic alterations generated during editing.
The work was led by SR-Tiget directors Luigi Nardini and Samuele Ferrari, with Daniele Canalutto and Martina Fiumara as first authors.
CRISPR-Cas9 editing has revolutionized the field of medical genetics by enabling targeted modification of disease-causing genes. The first CRISPR-based treatments, such as Exagam Glogen Autotemcell (Kasgebi) for sickle cell disease and transfusion-dependent beta-thalassemia, have already received regulatory approval in multiple countries, marking historic milestones in genome editing therapies. However, despite these advances, safety concerns remain. When CRISPR-Cas9 cuts DNA, cells can repair the break in unintended ways, resulting in chromosomal abnormalities and rearrangements that may carry unknown risks in terms of safety.
In addition, Casgevy is a drug based on the knockout of targeted genes through delivered DNA breaks. Conversely, targeted incorporation of DNA cassettes into cleavages has hitherto been out of reach due to limited efficiency as the results of targeted incorporation are inconsistent with other editing results. SMArT solves this problem by validating that the intended result has occurred, which enables enrichment of cells to 100% purity through targeted incorporation. Importantly, this increased efficiency also improves the safety profile.
These unintended consequences, such as large deletions of DNA sequences, have emerged as one of the most important limitations to the widespread application of gene editing, especially in stem cells for transplantation purposes. Using SMArT, we aimed to create an intelligent selection system that could identify and enrich only cells that achieved the desired genetic correction, while eliminating cells with potentially dangerous changes. ”
Luigi Nardini, San Raffaele Telethon Gene Therapy Institute
The researchers developed three increasingly sophisticated SMArT configurations that act as temporary synthetic “AND gate” systems. Only cells that simultaneously perform the intended on-target integration and maintain the integrity of the target locus will temporarily activate the selection marker, allowing purification of the correctly edited population.
In preclinical models, SMArT enrichment generated highly pure populations of edited blood stem cells while significantly reducing the presence of large deletions and other undesirable editing consequences. After transplantation into immunodeficient mice, the selected cells successfully engrafted and generated long-lasting human hematopoiesis. Importantly, the selectors used to isolate correctly edited cells are only expressed transiently and become undetectable after engraftment, leaving a “clean” edited graft.
“Our goal was not simply to improve editing efficiency, but to fundamentally rethink how we control the quality of edited cell products,” said Samuele Ferrari, co-senior author of the study. “SMArT introduces a programmable framework that can increase precision, reduce genotoxic burden, and preserve the functional potential of stem cells.”
Although this study focuses on therapeutic gene editing strategies for severe inherited immune diseases such as X-linked severe combined immunodeficiency (SCID-X1) and hyper-IgM 1 syndrome, the authors believe this approach has the potential to be broadly applicable across multiple gene editing platforms and disease areas.
One of the most advanced versions of this technology, SMArT-3, utilizes a single multifunctional, programmable CRISPR-based control system to transiently detect correct genomic integration and temporarily activate endogenous genes that have the potential to improve stem cell engraftment.
“Gene editing is often described as precision genome surgery, but the biology is more complex than initially expected,” said Daniele Canalutto, co-lead author of the study. “SMArT helps distinguish between cells that have truly achieved the intended therapeutic outcome and those that have undergone a different repair process.”
“Precision medicine requires precision editing,” added co-lead author Martina Fiumara. “We believe that approaches like SMArT can help us realize the full therapeutic potential of gene size editing while addressing some of the most pressing safety concerns in this field.”
The authors suggest that the SMArT strategy could be integrated not only with current CRISPR-Cas9 editing approaches, but also with other emerging genome engineering technologies.
This research was supported by the Fondazione Telethon, the European Union Horizon Europe Programme, the Italian Ministry of Health, the Italian Ministry of University and Research, and other international funding bodies.
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Reference magazines:
Kanalut, D. others. (2026). Selection of human hematopoietic stem cells with intended functional editing by a transient AND-gated reporter. nature biotechnology. DOI: 10.1038/s41587-026-03142-z. https://www.nature.com/articles/s41587-026-03142-z
