EFFICIENCY, FREQUENCY AND TIMING OF CRISPR-MEDIATED MUTAGENESIS IN ALFALFA

Targeted mutagenesis by CRISPR-Cas9 is particularly useful to edit agronomic traits of crops with complex genomes, such as polyploid species, because it enables precise targeting of multiple genes simultaneously. We targeted the glutamate-1-semialdehyde aminotransferase (GSA) gene to assess the efficiency of editing in terms of the percentage of edited transgenic events, type and frequency of edited alleles within events, presence of continued editing during plant development, and segregation of mutations from introduced sequences in sexual progeny. In this methodological work, Agrobacterium-mediated genetic transformation was used to introduce spCas9 and a guide RNA targeting the GSA gene into the M. sativa genotype RSY1. Nine transgenic events (E0) were obtained, and these were screened for mutations induced in the target site. E0 plants were crossed with a wildtype plant to obtain E1 progenies. Clonal progeny from selected E0 events were also produced. Genotyping of targeted gene mutations induced by CRISPR/Cas9 was then assayed in the E0 lines, their clonal progenies and the sexually derived E1 progeny. Preliminary TIDE (Tracking of Indels by Decomposition) analysis indicated a 44% success rate, with 4 edited events obtained. Sanger sequencing of about 20 cloned fragments per event revealed that all TIDE-positive events were edited, with varying frequencies of mutated (indel) and wild-type alleles, except for one event, that was completely edited and heterozygous with three alleles. A previously undetected allele was identified, likely involved in the homology-directed repair of double-strand breaks at the target site. Analysis of ten E1 progenies per event by PCR for transgene segregation demonstrated the possibility of obtaining non-transgenic edited progenies. Illumina Amplicon Sequencing of the target site was performed to comprehensively assess mutation efficiency, edited allele percentage, allelic diversity, and Cas9/gRNA activity during sexual and asexual reproduction. This work provides useful data on the patterns and outcomes of CRISPR-Cas9 gene editing in an important tetraploid forage crop. This work was partly funded by the “Progetto Ricerca di Base”, Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università degli Studi di Perugia and by Agritech National Research Center under the European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA, PNRR – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4—D.D. 1032 17/06/2022, CN00000022)