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.
Highlights
Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing
We demonstrated that maize genes could be mutagenized and edited using Cas[9] and guide RNA (gRNA) delivered on DNA vectors[11,20]
Embryos bombarded with Cas[9] protein alone were used as negative controls, while DNA vectors encoding Cas[9] and the four gRNAs served as positive controls
Summary
Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. 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 demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. DNA molecules often integrate into the targeted DSB sites, decreasing the efficiency of gene editing and gene insertion[11,12] To mitigate these negative effects, plants with pre-integrated Cas[9] nuclease have been generated and used for delivery of gRNA in the form of RNA molecules[11]. The authors demonstrated efficient gene mutagenesis in plants using transiently expressed CRISPR–Cas[9] DNA and by delivering Cas[9] and gRNA as in vitro transcribed RNA molecules
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