Viable mutants of essential genes in Physcomitrium patens as tools for studying primary metabolic processes

Abstract

Sphingolipids are essential components of plant cells, which have been notoriously difficult to study in part due to pleiotropic or lethal knock-out mutant phenotypes. By relying on alternative end-joining of double stranded breaks, we successfully used CRISPR/Cas9 mutagenesis to generate a population of diverse, viable mutant alleles of genes required for sphingolipid assembly in totipotent protoplasts of the moss Physcomitrium patens. We targeted the INOSITOL PHOSPHORYLCERAMIDE SYNTHASE (IPCS) gene family, which catalyzes the committed step in the synthesis of glycosyl inositol phosphorylceramides (GIPCs), the most abundant class of sphingolipids found in plants. We isolated knock-out single mutants and knock-down higher-order mutants showing a spectrum of deficiencies in GIPC content. Remarkably, we also identified two mutant alleles accumulating inositol phosphorylceramides, the direct products of IPCS activity, and provide our best explanation for this unexpected phenotype. Our approach is broadly applicable for studying essential genes and gene families, and for obtaining unusual lesions within a gene of interest.