The Protease WSS1A, the Endonuclease MUS81, and the Phosphodiesterase TDP1 are Involved in Independent Pathways of DNA-protein Crosslink Repair in Plants
Janina Enderle, Annika Dorn, Natalja Beying, Oliver Trapp and Holger Puchta
DNA-protein crosslinks (DPCs) represent a severe threat to the genome integrity; however, the main mechanisms of DPC repair were only recently elucidated in humans and yeast. Here we define the pathways for DPC repair in plants. Using CRISPR/Cas9, we could show that only one of two homologues of the universal repair proteases SPRTN/Wss1, WSS1A, is essential for DPC repair in Arabidopsis thaliana. WSS1A defective lines exhibit developmental defects and are hypersensitive to camptothecin (CPT) and cis-platin. Interestingly, the CRISPR/Cas9 mutants of TYROSYL-DNA PHOSPHODIESTERASE 1 (TDP1) are insensitive to CPT, and only the wss1A tdp1 double mutant reveals a higher sensitivity than the wss1A single mutant. This indicates that TDP1 defines a minor backup pathway in the repair of DPCs. Moreover, we found that knock out of the endonuclease MMS AND UV SENSITIVE PROTEIN 81 (MUS81) results in a strong sensitivity to DPC-inducing agents. The fact that wss1A mus81 and tdp1 mus81 double mutants exhibit growth defects and an increase in dead cells in root meristems after CPT treatment, demonstrates that there are three independent pathways for DPC repair in Arabidopsis. These pathways are defined by their 35 heir different biochemical specificities, as main actors, the DNA endonuclease MUS81 and the protease WSS1A, and the phosphodiesterase TDP1 as backup.
Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system
Carla Schmidt, Michael Pacher and Holger Puchta
During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Thus, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from S. aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild-type and in the non-homologous end joining (NHEJ) mutant ku70-1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild-type, most junctions are formed by microhomology-mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg cell specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will.
The topoisomerase 3α zinc-finger domain T1 of Arabidopsis thaliana is required for targeting the enzyme activity to Holliday junction-like DNA repair intermediates
Annika Dorn, Sarah Röhrig, Kristin Papp, Susan Schröpfer, Frank Hartung, Alexander Knoll, Holger Puchta
DNA Topoisomerases are essential for transcription, DNA repair and DNA replication
due to their function to break and change the topological state of DNA molecules. Topoisomerase 3α is especially important for DNA repair as it is able to process different DNA recombination and repair intermediates. The protein can be divided into three different functional domains, the TOPRIM domain, the active centre and the zinc-finger domains (ZFDs). Whereas the first two domains are essential for the basic functions of DNA binding and breaking, the role of the ZFDs was elusive until now. By deletion analysis and complementation studies in the model plant Arabidopsis thaliana, we were now able to show that ZFDs are required to target the topoisomerase to specific DNA structures that arise during the repair of aberrant DNA replication intermediates, the Holliday Junctions (HJs). We were able to demonstrate, by expressing a bacterial HJ resolvase in plant cells, that Topoisomerase 3α can only process these intermediates when the ZFDs are part of the protein. However, in the case of a non-functional topoisomerase, the presence of the ZFDs leads to masking of these HJs so that they cannot be processed by other plant nucleases, leading to cell death.
Efficient in planta gene targeting in Arabidopsis using egg-cell specific expression of the Cas9 nuclease of S. aureus
Felix Wolter, Jeanette Klemm and Holger Puchta
Gene targeting (GT), the programmed change of genomic sequences by homologous recombination (HR), is still a major challenge in plants. We previously developed an in planta GT strategy by simultaneously releasing from the genome a dsDNA donor molecule and creating a DSB at a specific site within the targeted gene. Using Cas9 form S. pyogenes (SpCas9) under the control of a ubiquitin gene promoter, we obtained seeds harbouring GT events, although at a low frequency. In the present research we tested different developmentally controlled promotors and different kinds of DNA lesions for their ability to enhance GT of the acetolactate synthase (ALS) gene of Arabidopsis. For this purpose, we used the S. aureus Cas9 (SaCas9) nuclease and the SpCas9 nickase in various combinations. Thus, we analysed the effect of single strand break (SSB) activation of a targeted gene and/or the HR donor region. Moreover, we tested whether DSBs with 5’ or 3’ overhangs can improve in planta GT. Interestingly, the use of the SaCas9 nuclease controlled by an egg cell specific promoter was the most efficient: depending on the line, in the very best case 6% of all seeds carried GT events. In a third of all lines, the targeting occurred around the one percent range of the tested seeds. Molecular analysis revealed that in about half of the cases perfect HR of both DSB ends occurred. Thus, using the improved technology, it should now be feasible to introduce any directed change into the Arabidopsis genome at will. in vivo.