Abstract
The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by site-specific nucleases to initiate DNA repair reactions that are either based on non-homologous end joining (NHEJ) or homologous recombination (HR). Recently, the CRISPR/Cas system emerged as the most important tool for genome engineering due to its simple structure and its applicability to a wide range of organisms. Here, we review the current status of its various applications in plants, where it is used for the successful generation of stable mutations in a steadily growing number of species through NHEJ. Furthermore, tremendous progress in plant genome engineering by HR was obtained by the setup of replicon mediated and in planta gene targeting techniques. Finally, other complex approaches that rely on the induction of more than one DNA lesion at a time such as paired nickases to avoid off-site effects or controlled genomic deletions are beginning to be applied routinely.
Highlights
With the rise of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated) technology double-strand break (DSB)-induced genome engineering moved into the centre of scientific interest in the last 2 years, the basic principles behind this approach were well known for decades
Background with the rise of the CRISPR/Cas technology double-strand break (DSB)-induced genome engineering moved into the centre of scientific interest in the last 2 years, the basic principles behind this approach were well known for decades
Scientists from UCSD recently demonstrated that a putative key player of auxin signalling in Arabidopsis (ABP1) does not possess the important function that was inferred from the analysis of plants obtained by more classical mutagenesis techniques [60]
Summary
With the rise of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated) technology double-strand break (DSB)-induced genome engineering moved into the centre of scientific interest in the last 2 years, the basic principles behind this approach were well known for decades. When the constructs were applied to the HR reporter plant lines DGU.US and IU.GUS [28], the nickase was able to induce HR at least as efficiently as the Cas9 nuclease, rendering it a promising tool for HRbased genome engineering approaches.
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