Yaoyao Lu

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, a genome-editing technology pioneered in 2012, enables the precise correction of deleterious mutations or disruption of disease-causing genes through targeted double-strand breaks (DSBs), offering potential for treating genetic diseases. However, CRISPR/Cas9 can cause off-target cleavage at non-specific DNA sites, leading to unintended insertions or deletions (indels), which limit its safety and applicability despite ongoing improvements in specificity. Recently, prime editing (PE), an advanced CRISPR-derived technology, has been employed with a Cas9 nickase (Cas9n) fused with a reverse transcriptase and a prime editing guide RNA (pegRNA) to enable precise insertions, deletions, and transversions without inducing DSBs, thus reducing risks of indels and chromosomal aberrations. Furthermore, ongoing optimizations, such as improved pegRNA design and enhanced editing efficiency, have expanded the applications of PE in medical therapeutics, agriculture, and fundamental research. This review summarizes recent advancements in the PE system, including optimized pegRNA designs and enzyme engineering for enhanced efficiency and specificity, alongside novel delivery methods. It also evaluates cutting-edge delivery strategies, such as adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs) and novel extracellular vesicle (EV)-based systems, and explores PE applications in vitro and in vivo, including disease modeling and therapeutic gene correction.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an exceptionally potent genome-editing technique developed in 2012, is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. However, it is prone to cleaving double-stranded DNA in off-target genes and generating random mutations in the process. These drawbacks restrict its application in fundamental research and agriculture, and raises safety concerns in the field of medicine. Fortunately, the new gene editing technology derived from CRISPR/Cas9, known as prime editing, has the potential to provide targeted sequence insertion, deletion, and transversion, all while avoiding the formation of double-strand breaks, thus minimizing adverse effects. Meanwhile, the rapid development of this technology makes its application wider and broader. This review summarizes the current developments and optimizations of the prime editing (PE) system with improved editing efficiency and precision. Along with discussing the most recent delivery techniques and outlining the PE applications that are being used both in vitro and in vivo.