Natural and artificial evolution of acetolactate synthase for crop breeding

Acetolactate synthase (ALS)-targeting herbicides are among the most widely used weed-control chemicals globally. Mutations in the ALS gene can confer herbicide resistance in crops, thereby allowing selective elimination of weeds without harming crops. Herbicide-resistant ALS alleles were initially discovered in weeds and subsequently developed through artificial mutagenesis techniques. With the advancement of CRISPR/Cas technologies, various genome-editing tools are now available to introduce these resistant alleles, as well as novel variants, into diverse crop species. Moreover, emerging methodologies, such as directed evolution, enable the generation and screening of large populations of random ALS mutants. Consequently, ALS has become one of the most extensively targeted genes in plant gene evolution. This paper provides a comprehensive overview of both conventional and recently developed strategies for ALS evolution, with particular emphasis on CRISPR/Cas-based genome editing and directed evolution. Future perspectives on technological application are also discussed. By advancing our understanding of herbicide-resistant ALS allele development for crop improvement, these methodologies may also pave the way for their application to the evolution of other agronomically important genes.