Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize

Recent advances in the development of CRISPR-Cas genome editing technologies have made it possible to perform targeted mutagenesis and precise gene replacement in crop plants. CRISPR-Cas9 and CRISPR-Cas12a are two main types of widely used genome editing systems. However, when CRISPR-Cas12a editing machinery is expressed from a transgene, some chromosomal targets encountered low editing frequency in important crops like maize and soybean. Here, we report efficient methods to directly generate genome edited lines by delivering Cas12a-gRNA ribonucleoprotein complex (RNP) to immature maize embryos through particle bombardment in an elite maize variety. Genome edited lines were obtained at ~7% frequency without any selection during regeneration via biolistic delivery of Cas12a RNP into immature embryos. Strikingly, the gene editing rate was increased to 60% on average and up to 100% in some experiments when the Cas12a RNP was co-delivered with a PMI selectable marker gene cassette and the induced callus cultures were selected with mannose. We also show that use of higher activity Cas12a mutants resulted in improved editing efficiency in more recalcitrant target sequence. The advances described here provide useful tools for genetic improvement of maize.

Sphingolipid Effects on the Plasma Membrane Produced by Addition of Fumonisin B1 to Maize Embryos

Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides that modifies the membrane properties from animal cells and inhibits complex sphingolipids synthesis through the inhibition of ceramide synthase. The aim of this work was to determine the effect of Fumonisin B1 on the plant plasma membrane when the mycotoxin was added to germinating maize embryos. Fumonisin B1 addition to the embryos diminished plasma membrane fluidity, increased electrolyte leakage, caused a 7-fold increase of sphinganine and a small decrease in glucosylceramide in the plasma membrane, without affecting phytosphingosine levels or fatty acid composition. A 20%–30% inhibition of the plasma membrane H+-ATPase activity was observed when embryos were germinated in the presence of the mycotoxin. Such inhibition was only associated to the decrease in glucosylceramide and the addition of exogenous ceramide to the embryos relieved the inhibition of Fumonisin B1. These results indicate that exposure of the maize embryos for 24 h to Fumonisin B1 allowed the mycotoxin to target ceramide synthase at the endoplasmic reticulum, eliciting an imbalance of endogenous sphingolipids. The latter disrupted membrane properties and inhibited the plasma membrane H+-ATPase activity. Altogether, these results illustrate the mode of action of the pathogen and a plant defense strategy.