Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes

Abstract

Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR–Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9–gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species.

Abstract

Genome editing in plants typically requires the expression of Cas9 and guide RNA from stably transformed plasmid DNA. Here, the authors show that successful editing can be achieved after delivery of the Cas9-guide RNA complex as a ribonucleoprotein to maize embryos via biolistics.