Abstract
The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.
Highlights
Our ability to knock out genes in diverse eukaryotic species has dramatically increased due to advances in the development of site-specific nucleases (Kim and Kim, 2014; Voytas and Gao, 2014; Osakabe and Osakabe, 2015)
The RNA templates were generated as a cis repair template, where the single-guide RNA (sgRNA) and the repair template work as a single bifunctional molecule (Figure 1A), or as a trans repair template, where the sgRNA and the repair template were separated by a tRNA, which was further processed to give two RNA molecules (Figure 1B) (Xie et al, 2015)
Two different scaffold sgRNAs were tested during the cis repair, the scaffold RNA sequence used for rice (Xie et al, 2015) and the human scaffold RNA sequence as previously described (Chen et al, 2013; Zalatan et al, 2015)
Summary
Our ability to knock out genes in diverse eukaryotic species has dramatically increased due to advances in the development of site-specific nucleases (Kim and Kim, 2014; Voytas and Gao, 2014; Osakabe and Osakabe, 2015). Engineering of a 20-nucleotide sequence in single or multiple sgRNAs, for single or multiple targets, respectively, has facilitated efficient genome engineering in transformable eukaryotic species (Gasiunas et al, 2012; Cong et al, 2013; DiCarlo et al, 2013; Li et al, 2013; Mali et al, 2013; Nekrasov et al, 2013; Aouida et al, 2015). The CRISPR/Cas system generates site-specific double strand breaks (DSBs) at userdefined genomic sequences, which are repaired mainly through the error-prone non-homologous end joining (NHEJ) process or the more precise homology-directed repair (HDR) process
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