An inducible CRISPR-Kill system for temporally controlled cell type-specific cell ablation in Arabidopsis thaliana.

The application of the CRISPR/Cas system as a biotechnological tool for genome editing has revolutionized plant biology. Recently, the repertoire was expanded by CRISPR-Kill, enabling CRISPR/Cas-mediated tissue engineering through genome elimination by tissue-specific expression. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), CRISPR-Kill relies on the induction of multiple double-strand breaks (DSBs) in conserved repetitive genome regions, such as the rDNA, causing cell death of the targeted cells. Here, we show that in addition to spatial control by tissue-specific expression, temporal control of CRISPR-mediated cell death is feasible in Arabidopsis thaliana. We established a chemically inducible tissue-specific CRISPR-Kill system that allows the simultaneous detection of targeted cells by fluorescence markers. As proof of concept, we were able to eliminate lateral roots and ablate root stem cells. Moreover, using a multi-tissue promoter, we induced targeted cell death at defined time points in different organs at select developmental stages. Thus, using this system makes it possible to gain new insights into the developmental plasticity of certain cell types. In addition to enabling tissue engineering in plants, our system provides an invaluable tool to study the response of developing plant tissue to cell elimination through positional signaling and cell-to-cell communication.

Nonhomologous end joining as key to CRISPR/Cas-mediated plant chromosome engineering.

Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-mediated gene editing has revolutionized biology and plant breeding, large-scale, heritable restructuring of plant chromosomes is still in its infancy. Duplications and inversions within a chromosome, and also translocations between chromosomes, can now be achieved. Subsequently, genetic linkages can be broken or can be newly created. Also, the order of genes on a chromosome can be changed. While natural chromosomal recombination occurs by homologous recombination during meiosis, CRISPR/Cas-mediated chromosomal rearrangements can be obtained best by harnessing nonhomologous end joining (NHEJ) pathways in somatic cells. NHEJ can be subdivided into the classical (cNHEJ) and alternative NHEJ (aNHEJ) pathways, which partially operate antagonistically. The cNHEJ pathway not only protects broken DNA ends from degradation but also suppresses the joining of previously unlinked broken ends. Hence, in the absence of cNHEJ, more inversions or translocations can be obtained which can be ascribed to the unrestricted use of the aNHEJ pathway for double-strand break (DSB) repair. In contrast to inversions or translocations, short tandem duplications can be produced by paired single-strand breaks via a Cas9 nickase. Interestingly, the cNHEJ pathway is essential for these kinds of duplications, whereas aNHEJ is required for patch insertions that can also be formed during DSB repair. As chromosome engineering has not only been accomplished in the model plant Arabidopsis (Arabidopsis thaliana) but also in the crop maize (Zea mays), we expect that this technology will soon transform the breeding process.

Protocol to efficiently induce CRISPR-Kill-mediated cell ablation in Arabidopsis thaliana.

The CRISPR-Kill system enables targeted cell ablation by inducing multiple double-strand breaks in evolutionarily conserved repetitive genomic regions. Here, we present a protocol for the application of the CRISPR-Kill system to analyze the systemic and cellular effects of targeted cell death in Arabidopsis. We describe steps for generating constitutive and inducible CRISPR-Kill lines, chemically inducing CRISPR-Cas9-mediated genome elimination, and monitoring of cell death in shoot and root apical meristems. This enables the investigation of a wide range of questions in developmental plant biology. For complete details on the use and execution of this protocol, please refer to Gehrke et al.1.

Using CRISPR-Kill for organ specific cell elimination by cleavage of tandem repeats.

CRISPR/Cas has been mainly used for mutagenesis through the induction of double strand breaks (DSBs) within unique protein-coding genes. Using the SaCas9 nuclease to induce multiple DSBs in functional repetitive DNA of Arabidopsis thaliana, we can now show that cell death can be induced in a controlled way. This approach, named CRISPR-Kill, can be used as tool for tissue engineering. By simply exchanging the constitutive promoter of SaCas9 with cell type-specific promoters, it is possible to block organogenesis in Arabidopsis. By AP1-specific expression of CRISPR-Kill, we are able to restore the apetala1 phenotype and to specifically eliminate petals. In addition, by expressing CRISPR-Kill in root-specific pericycle cells, we are able to dramatically reduce the number and the length of lateral roots. In the future, the application of CRISPR-Kill may not only help to control development but could also be used to change the biochemical properties of plants.