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. 2016 Nov 16;7:13274.
doi: 10.1038/ncomms13274.

Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes

Sergei Svitashev  1 , Christine Schwartz  1 , Brian Lenderts  1 , Joshua K Young  1 , A Mark Cigan  1
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Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes

Sergei Svitashev et al. Nat Commun. .
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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.

Figures

Figure 1. Examples of four of the most prevalent mutation types at four targeted sites generated by delivery of Cas9 and gRNAs in the form of RNPs or DNA vectors.
LIG, ALS2, MS26 and MS45 were targeted. Protospacer adjacent motif (PAM) sequences are underlined and mutations are indicated as red dashes or letters. Black dashes indicate spaces for alignment purposes. WT indicates wild-type sequences.
Figure 2. ALS2 gene editing using co-delivery of RNP complex and single-stranded (ss) oligo as repair template.
(a) Partial ALS2 sequence with ALS–gRNA target site, protospacer adjacent motif (PAM) is boxed and proline codon (amino acid position 165) to be edited is underlined. (b) Partial sequence of the ssDNA oligo used as a repair DNA template; modified nucleotides are shown in red font and serine codon is underlined. (c) Wild type (left) and ALS2 edited (right) plants tested for resistance to chlorsulfuron (200 mg l−1); shown at 10 days after spraying.
Figure 3. Biallelic mutations in MS45 by RNP result in male sterile maize.
(a) Male-fertile tassel of wild-type maize. (b) Male-sterile tassel of biallelic ms45 mutant.
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