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.
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
Sarah Röhrig, Annika Dorn, Janina Enderle, Angelina Schindele, Natalie J. Herrmann, Alexander Knoll and Holger Puchta
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.in vivo.
Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes
Simon Schiml, Friedrich Fauser and Holger Puchta