Dem IPB wird erneut ein beispielhaftes Handeln im Sinne einer chancengleichheitsorientierten Personal- und Organisationspolitik bescheinigt. Das Institut erhält zum 6. Mal in Folge das TOTAL E-QUALITY…
Die Plant Science Student Conference (PSSC) wird seit 20 Jahren im jährlichen Wechsel von Studierenden der beiden Leibniz-Institute IPK und IPB organisiert. Im Interview erläutern Christina Wäsch…
The recent discovery of the mode of action of the CRISPR/Cas9 system has
provided biologists with a useful tool for generating site-specific
mutations in genes of interest. In plants, site-targeted mutations are
usually obtained by the stable transformation of a Cas9 expression
construct into the plant genome. The efficiency of introducing mutations
in genes of interest can vary considerably depending on the specific
features of the constructs, including the source and nature of the
promoters and terminators used for the expression of the Cas9 gene and
the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize
the efficiency of the Cas9 nuclease in generating mutations in target
genes in Arabidopsis thaliana, we investigated several features
of its nucleotide and/or amino acid sequence, including the codon
usage, the number of nuclear localization signals (NLSs), and the
presence or absence of introns. We found that the Cas9 gene codon usage
had some effect on its activity and that two NLSs worked better than
one. However, the highest efficiency of the constructs was achieved by
the addition of 13 introns into the Cas9 coding sequence, which
dramatically improved the editing efficiency of the constructs. None of
the primary transformants obtained with a Cas9 gene lacking introns
displayed a knockout mutant phenotype, whereas between 70% and 100% of
the primary transformants generated with the intronized Cas9 gene
displayed mutant phenotypes. The intronized Cas9 gene was also found to
be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.