Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas

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.

Keywords: Double-strand break repair; Gene targeting; Gene technology; Synthetic nucleases; Targeted mutagenesis.

Fig. 1

Schematic representation of the Cas9 cleavage mechanism. The Cas9/sgRNA complex recognizes and binds the complementary sequence next to the PAM, which is highly specific for each Cas9 from different bacterial species. The RuvC domain and HNH motif of Cas9 cleave the two DNA strands 3 bp upstream of the PAM

Fig. 2

Cas9 paired nickases approach. By using 2 sgRNAs, the D10A nickase variant can be guided to the two opposite DNA strands at adjacent positions. The resulting BSD exhibits long single-stranded 5′-overhangs

Fig. 3

Overview of the Cas9-mediated in planta GT system. The nuclease and the DNA donor sequence are located on one T-DNA that is stably transformed into the plant. The nuclease induces two DSBs that release the donor intermediate and a third DSB that activates the target locus for HR. The donor sequence integrates into the target locus by using the flanking homologous regions

Fig. 4

Replicon-mediated GT. The replicon is released from the T-DNA, circularises at the LIRs and undergoes rolling circle replication. This leads to the promoter being positioned upstream of the nuclease ORF. Upon DSB-induction in the target locus, the integration of the donor sequence can be achieved by HR