Targeted insertion of large DNA fragments has been constrained by the genotoxicity associated with double-strand breaks (DSBs) and the limitations of two-step recombinase systems. Three independently developed mammalian platforms, Quadruple pegRNA (QuadPE), Prime Assembly (PA), and PRIME-In, now achieve programmable, DSB- and recombinase-free insertion of up to 26 kb, 11.3 kb, and 9.2 kb, respectively, through convergent yet mechanistically distinct flap-guided integration strategies. Notably, QuadPE and PA achieve integration independent of cell-cycle progression. Furthermore, these systems outperform several existing recombinase- and/or transposase-based tools, underscoring their translational potential for therapeutic utility. Their mechanistic logic also provides a forward path for the DSB-free and recombinase-free integration of large DNA sequences in crop biotechnology.
Excessive intake of free sugars is a major modifiable risk factor for obesity, type 2 diabetes and cardiovascular disease, and it is a global public-health priority to reduce dietary sugar while preserving sensory quality. Referred to here as “healthy sugars”, the low-calorie monosaccharides D-allulose and D-tagatose combine sucrose-like sensory and bulking behavior with a distinct metabolic fate in humans and, for D-tagatose, with defence-inducing activity in plants. This review traces the evolution of sugar supply through four stages, from agricultural extraction of sucrose (Sugar 1.0) and enzymatic hydrolysis to high-fructose syrup (Sugar 2.0) to chemically synthesized high-intensity sweeteners (Sugar 3.0) and precision biomanufacturing of healthy sugars (Sugar 4.0). We review the molecular basis of healthy-sugar action in mammalian metabolic homeostasis and in plant sweet immunity, then compare manufacturing platforms, equilibrium-limited enzymatic epimerization, immobilized-enzyme and continuous-reactor processes, thermodynamically driven cell-free cascades, and cell-free CO2-to-hexose synthesis, through a consolidated cross-platform comparison of conversion, titer, yield, scalability and technology readiness. We then set out an amyloplast-targeted compartmentalised design for sugar-crop chassis, a distinct phosphorylated route for D-tagatose, and a climate-adaptive sugarcane, sugar beet, sorghum manufacturing crops. Finally we examine the conditions under which a plant-chassis route could offer cost or carbon advantages, comparing feedstock, energy, separation and carbon-balance considerations across platforms, and comparing the ingredient and biosafety requirements applying in the United States, the European Union and China. The plant-chassis route is presented throughout as a design proposal requiring crop-level validation, and the prerequisites for its projected advantages are stated explicitly.
