Scientists from Institut National de la Recherche Agronomique in France
together with partners in Italy reported single and multiple gene
mutagenesis using stably transformed plants. The findings are published in
Plant Cell Reports.
The team used two different CRISPR-Cas9 vectors allowing the expression of
multiple guide RNAs and various techniques to knockout either independent or
paralogous genes. They generated 12 plasmids that represent 28 various
single guide RNAs (sgRNAs) to target 20 genes. For 18 of the target genes,
at least one mutant allele was obtained, while two genes were recalcitrant
to sequence editing.
It was observed that there were small insertions or deletions of less than
ten nucleotides, regardless of whether the gene was targeted by one or more
sgRNAs. They also found deletions of defined regions located between the
target sites of two guide RNAs. Furthermore, double and triple mutants were
created in a single step, which is important for functional analysis of
genes with strong genetic linkage. Tests also showed that the majority (85%)
were fully edited plants transmitting systematically all detected mutations
to the next generation, generally following Mendelian segregation.