Transgene-free genome editing in plants.

Genome-editing tools for the precise, efficient modification of DNA have led to groundbreaking advances in crop improvement and basic plant science research. However, conventional genome editing may result in the integration of unintended gene fragments into the host genome, along with off-target effects and the risk of genetic drift of resistance genes. By contrast, transgene-free genome editing represents a revolutionary breakthrough due to its ability to 1) achieve precise and stable genomic modifications while minimizing foreign DNA integration through DNA-free or transient delivery of CRISPR components; and 2) deliver ribonucleoprotein complexes (RNPs) or mRNA into the host. In this review, we discuss the core principles of transgene-free genome-editing technologies, including the direct delivery of RNPs, mRNA delivery systems, the precise substitution of single nucleotides using cytosine or adenine base editors (CBEs, ABEs), and the mechanisms behind multiple types of prime editing that use templates for reverse transcriptases. These techniques have driven the development of crops considered nongenetically modified, as they do not contain stably integrated foreign DNA. Finally, we discuss future prospects, including the development of transgene-free genome-editing tools that combine delivery systems with artificial intelligence-assisted optimization, with promising applications for agriculture.