LbCas12-mediated multiplex gene editing and 2-fluoroadenine counter-selection in Phytophthora palmivora

CRISPR-Cas systems have moved forward genetic engineering in virtually any organism amenable to genetic modification. In particular, these systems have unlocked unprecedented possibilities to generate mutants in oomycetes, a group of filamentous microbes comprising over two hundred Phytophthora species, including the cacao killer Phytophthora palmivora. Here, we showcase multiplex gene editing in P. palmivora using LbCas12. We have developed a straightforward protocol to simultaneously knock out two genes encoding adenine phosphoribosyltransferase (APT), an essential enzyme of the purine salvage pathway. We show that APT knockouts ({Delta}PpATP1/2) are insensitive to 2-fluoroadenine (2-FA) and retain full virulence on Nicotiana benthamiana. We rely on zoospore electroporation using an all-in-one construct to facilitate the rapid editing of multiple genes. This work enhances the genetic toolbox for Phytophthora species and simplifies the exploration of gene function, laying the groundwork for future innovations aiming to tackle oomycete plant diseases.