Comprehensive analysis of bZip transcription factors using CRISPR-mediated foreign DNA incorporations in the tomato wilt fungus

Transient expression of the CRISPR system and the induction of DNA cleavage are effective for homologous recombination-mediated genome editing in many filamentous fungi.Meanwhile, constitutive expression of CRISPR system often fails to induce mutations.In the present study, we discovered that CRISPR-mediated target gene cleavage induces exogenous DNA degradation and incorporation into the genome of the tomato wilt fungus Fusarium oxysporum f. sp.lycopersici (Fol).Based on this mechanism, we constructed a mutant library of bZip transcription factors (53 targets) and performed a comprehensive analysis of their functions.A CRISPR library targeting 53 genes was created, and transformants were obtained by introducing them.PCR analyses showed that 19 out of the 53 genes were disrupted by multiple integrations of the DNA fragments of the delivered CRISPR plasmid into each cleavage site.To improve the plasmid, a gRNA targeting the CRISPR vector sequence was additionally inserted into the plasmid, which can simultaneously induce DNA cleavage at both the delivered plasmid and the target gene in the nucleus.The improved vector elevated the efficiency, and 40 mutants were obtained.Phenotypic analyses revealed that several mutants exhibited significantly reduced growth and virulence to tomato plants.Meanwhile, the growth and pathogenicity of most of the mutants did not change, suggesting that the integration of CRISPR vector fragments did not affect the phenotypes.