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.

The RecQ-like helicase HRQ1 is involved in DNA crosslink repair in Arabidopsis in a common pathway with the Fanconi anemia-associated nuclease FAN1 and the postreplicative repair ATPase RAD5A.

RecQ helicases are important caretakers of genome stability and occur in varying copy numbers in different eukaryotes. Subsets of RecQ paralogs are involved in DNA crosslink (CL) repair. The orthologs of AtRECQ2, AtRECQ3 and AtHRQ1, HsWRN, DmRECQ5 and ScHRQ1 participate in CL repair in their respective organisms, and we aimed to define the function of these helicases for plants. We obtained Arabidopsis mutants of the three RecQ helicases and determined their sensitivity against CL agents in single- and double-mutant analyses. Only Athrq1, but not Atrecq2 and Atrecq3, mutants proved to be sensitive to intra- and interstrand crosslinking agents. AtHRQ1 is specifically involved in the repair of replicative damage induced by CL agents. It shares pathways with the Fanconi anemia-related endonuclease FAN1 but not with the endonuclease MUS81. Most surprisingly, AtHRQ1 is epistatic to the ATPase RAD5A for intra- as well as interstrand CL repair. We conclude that, as in fungi, AtHRQ1 has a conserved function in DNA excision repair. Additionally, HRQ1 not only shares pathways with the Fanconi anemia repair factors, but in contrast to fungi also seems to act in a common pathway with postreplicative DNA repair.

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.

CRISPR/Cas brings plant biology and breeding into the fast lane.

CRISPR/Cas is in the process of inducing the biggest transformation of plant breeding since the green revolution. Whereas initial efforts focused mainly on changing single traits by error prone non-homologous end joining, the last two years saw a tremendous technical progress achieving more complex genetic, epigenetic and transcriptional changes. The efficiencies of inducing directed changes by homologous recombination have been improved significantly and strategies to break genetic linkages by inducing chromosomal rearrangements have been developed. Cas13 systems have been applied to degrade viral and mRNA in plants. Most importantly, a historical breakthrough was accomplished: By introducing multiple genomic changes simultaneously, domestication of wild species in a single generation has been demonstrated, speeding up breeding dramatically.