Next-Generation Genome Editing: Overcoming CRISPR-Cas9 Limitations with Prime Editing and PASTE Technology
CRISPR-Cas9 has transformed gene editing; however, it is based on doublestrand DNA breaks, which may result in unintentional mutations and structural alterations, thereby limiting its clinical translation. Instead of DSBs, new precision instruments, including prime editing, can be used to perform accurate search-and-replace DNA editing with a Cas9 nickase and reverse transcriptase. Prime editing can perform all base substitutions and small edits with improved precision and reduced off-target activity, which has promising results in disease modelling with patient-derived organoids. Nonetheless, it is cumbersome, inefficient, and thus hard to deliver. This is improved by the Programmable Addition via Site-specific Targeting Elements system, which uses Prime editing in combination with serine integrases to insert large DNA sequences with no gaps to provide lasting gene replacement and maintain natural gene regulation. Emerging delivery options, including lipid nanoparticles, dual adeno-associated virus systems, and virus-like particles, allow the delivery to be transient and tissue-targeted, which minimizes the risks over the long-term. Early clinical translation efforts are currently underway, with the first FDA-approved trial of one Prime editing-based therapy, but it has been struggling with efficiency, safety, and delivery. Future success will depend on predictive machinelearning devices and strictly controlled editing systems. Genome engineering is evolving from a DNA-cutting system to a precise and programmable genome editing system, which may support the future development of safer and more precise therapeutic strategies.