Simple and efficient targeting of multiple genes through CRISPR-Cas9 in Physcomitrella patens

Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene knock-out-technology in a variety of species. However, in a number of situations knocking out or modifying a single gene is not sufficient, this is particularly true for genes belonging to a common family or for genes showing redundant functions. Like many plants the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the co-delivery of a CAS9 expressing cassette, multiple sgRNA vectors and a cassette for transient transformation selection for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9 mediated targeting of five different genes allows the selection of a quintuple mutant and all possible sub-combinations of mutants in one experiment with no mutations detected in potential off target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to alternative End Joining pathway for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants

Plant growth : the translational connection

International audienceThe TOR (target of rapamycin) pathway is a phylogenetically conserved transduction system in eukaryotes linking the energy status of the cell to the protein synthesis apparatus and to cell growth. The TOR protein is specifically inhibited by a rapamycin–FKBP12 complex (where FKBP stands for FK506-binding protein) in yeast and animal cells. Whereas plants appear insensitive to rapamycin, Arabidopsis thaliana harbours a single TOR gene, which is essential for embryonic development. It was found that the product of this gene was capable of binding to rapamycin and yeast FKBP12. In-frame fusion with a GUS reporter gene shows that the TOR protein is produced essentially in proliferating zones, whereas the TOR mRNA can be detected in all organs suggesting a translational regulation of TOR. Phenotypic analysis of Arabidopsis TOR mutants indicates that the plant TOR pathway fulfils the same role in controlling cell growth as its other eukaryotic counterparts