A streamlined protocol for small-scale protoplast generation and CRISPR/Cpf1-mediated genome editing in Fusarium oxysporum

Abstract

Fusarium oxysporum plays crucial roles as both a damaging plant pathogen and a model for studying host-pathogen interactions. It is well established that systematic functional genomics approaches are vital for advancing agricultural biotechnology and developing biological agents. Creating efficient gene-editing systems can help elucidate virulence mechanisms and enable rapid production of modified strains for practical use. However, current transformation methods face major challenges, such as high enzyme costs, and CRISPR/Cas9 systems are limited by PAM sequence availability and by blunt-end DNA cleavage, which hampers homologous recombination. Furthermore, complex guide RNA scaffolds complicate large-scale functional studies with traditional methods. To address these challenges, we have developed a streamlined CRISPR/Cpf1 (Cas12a) platform that combines small-scale protoplast preparation with significantly reduced enzyme use, exploiting Cpf1's unique features, such as flexible PAM recognition, staggered DNA cuts that promote recombination, improved target specificity, and simpler guide RNA design. This platform can also accelerate the development of biological agents and support high-throughput screening applications essential to the progress of agricultural biotechnology.