Since NHEJ is error-prone, small insertions or deletions (indels) of a few base pairs (bp) occur often at the DSB site, leading to frameshift mutations and gene knockouts (Pacher and Puchta, 2016).
(ii) An easier and more efficient approach is the use of the non-homologous end joining (NHEJ) repair pathway of the plant, which is the dominant repair pathway in most plants, such as Arabidopsis thaliana ( Arabidopsis). However, due to low integration rates of these sequences, HDR mediated gene editing in plants remains challenging. This homology directed repair (HDR) approach can be exploited to introduce specific sequences at the site of the DSB (Schiml et al., 2014 Baltes and Voytas, 2015). (i) DNA molecules with high homology to the DSB site can be used as repair template. Here, two main pathways can be distinguished (Salomon and Puchta, 1998). These DSBs are then repaired by the innate DNA repair mechanism of the plant cell. The system relies on the bacterial Cas9 nuclease from Streptococcus pyogenes (Cas9), which can be directed by a short artificial single guide RNA molecule (sgRNA) towards a genomic DNA sequence (Jinek et al., 2012), where it creates a double strand break (DSB). The CRISPR/Cas9 system (Cas9) provides a simple and widely applicable approach to modify genomic regions of interest and has therefore become the tool of choice for genome editing in plants and other organisms (Schiml and Puchta, 2016). Keywords: CRISPR/Cas9, Genome editing, Arabidopsis thaliana, Plants, Knockout
The procedure can likely be adapted for other transformable plant species. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond.
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