Efficient CRISPR/Cas9 Genome Editing in Alfalfa Using a Public Germplasm

BOTTERO, EMILIA; STRITZLER, MARGARITA; FRARE, ROMINA; AYUB, NICOLÁS; MASSA, GABRIELA; TAJIMA, HIROMI; FEINGOLD, SERGIO; SOTO, GABRIELA; GONZÁLEZ, MATÍAS; GÓMEZ, CRISTINA; BLUMWALD, EDUARDO

Frontiers in Agronomy

Frontiers

Año: 2021 vol. 3

2673-3218

Because its ability to acquire large amounts of nitrogen by symbiosis, tetraploid alfalfa isthe main source of vegetable proteins in meat and milk production systems in temperateregions. Alfalfa cultivation also adds fixed nitrogen to the soil, improving the productionof non-legumes in crop rotation and reducing the use of nitrogen fertilizers derived fromfossil fuel. Despite its economic and ecological relevance, alfalfa genetics remains poorlyunderstood, limiting the development of public elite germplasm. In this brief article, wereported the high-efficiency of alfalfa mutagenesis by using the public clone C23 andthe CRISPR/Cas9 system. Around half of the GUS overexpressing plants (35S-GUSunder C23 genomic background) transformed with an editing plasmid containing twosgRNAs against the GUS gene and the Cas9 nuclease exhibited absence of GUS activity.Nucleotide analysis showed that the inactivation of GUS in CRISPR/Cas9-editing eventswere produced via different modifications in the GUS gene, including frameshift andnon-sense mutations. Using the CRISPR/Cas9 system and two sgRNAs, we have alsoedited the alfalfa gene NOD26, generating plants with different doses of alleles at thislocus, including complete gene knockout at high efficiency (11%). Finally, we discuss thepotential applications of genome-editing technologies to polyploid research and to alfalfaimprovement public programs.