The newly expanded CAST platform, evoCAST, provides an unprecedented increase in gene editing efficiency and precision. Unlike current CRISPR-Cas9 methods that cut DNA and risk unwanted mutations, evoCAST lets us add therapeutic DNA to specific spots in the genome without breaking the DNA strands. 

"This is a major step forward for genome editing," stated lead author Isaac Witte. "With evoCAST, we can now insert entire genes with high accuracy and efficiency, paving the way for one-step, mutation-agnostic therapies for a wide range of inherited disorders." 

The innovation addresses a long-standing issue in gene therapy: the difficulty of transporting and inserting big DNA pieces, such as complete genes, into specific sections of the human genome. This skill is critical for treating loss-of-function genetic illnesses, which require the complete restoration of a gene rather than only its correction. 

Previously known CRISPR-associated transposases exhibited little activity in human cells, often less than 0.1%. The study team used a rigorous laboratory procedure known as phage-assisted continuous evolution (PACE) to evolve CAST components across hundreds of generations, greatly improving their performance. 

TnsB, a developed transposase protein, now has more than 200-fold greater activity in human cells than its native equivalent. When combined with additional designed components, the evoCAST system achieved gene insertion rates of 10%–30% in 14 different genomic targets in various human cell types, including disease-relevant "safe harbour" locations used in gene therapy. 

Surprisingly, evoCAST was able to deliver gene payloads greater than 10 kilobases with near-perfect precision. The inserted sequences had no detectable insertions or deletions (indels), little off-target effects, and a single base-pair resolution—features that considerably reduce safety risks in clinical settings. 

"The high precision and efficiency of evoCAST offer a new level of control in genome editing," Dr Liu added. "This tool has the potential to fundamentally change how we approach genetic disease treatment and cell engineering." 

Beyond genetic illness therapy, the researchers see potential uses in cancer immunotherapy, regenerative medicine, and the development of animal models for biomedical research. EvoCAST's compatibility with various human cell types, including primary fibroblasts, demonstrates its wide applicability. 

Additionally, this method does not use ClpX, a bacterial helper protein that was needed for CAST to work but can be harmful to human cells, making it safer for treatment use. 

According to the study, evoCAST could provide a "single-drug" solution for many genetically heterogeneous disorders by allowing the insertion of healthy gene copies rather than necessitating the development of several mutation-specific therapies. 

Equally important is the PACE-based evolution platform used to develop evoCAST. This approach enables rapid, guided development of gene editing tools, which might be used to improve CAST systems or adapt them to other complicated biological contexts. 

"This evolution framework is a powerful engine for discovery," wrote co-author George Lampe. "It enables us to continuously improve genome editing technologies, keeping pace with the growing complexity of biomedical challenges." 

The introduction of evoCAST represents a major milestone in genome editing technology. Researchers have developed a flexible and very precise tool by merging CRISPR's ability to be programmed with the insertion skills of transposases and improving their effectiveness through ongoing evolution. Genetic therapy could soon employ this technology. 

As gene therapy advances, breakthroughs like evoCAST bring us closer to a future in which once-incurable genetic illnesses can be treated with a single, tailored intervention.