Study Shows Single and Multiple Gene Knockouts by CRISPR-Cas9 in Maize

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.

Read the research article in Plant Cell Reports.