CRISPR/Cas9 gene editing in peanut by Agrobacterium tumefaciens -mediated pollen tube transformation

Peanut ( Arachis hypogaea L.) is an important global food crop, providing a valuable source of protein, oil and nutrition. The functional analysis of genes associated with yield traits is an important step in molecular breeding, which requires effective methods of genetic transformation. In this study, we applied the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system in conjunction with Agrobacterium tumefaciens -mediated pollen tube transformation to create knockout mutants of the peanut MUTATOR-LIKE TRANSPOSABLE ELEMENT9A ( AhMULE9A ) gene, which plays an importantly regulatory role in response to Al stress in peanut. The gene mutations were identified by the analysis of genomic sequences flanking the sgRNA target sites. The sequencing of AhMULE9A mutations showed that eleven of the fifteen transgenics produced resulted in protein sequence changes, demonstrating the efficacy of this method for the production of targeted mutations in allotetraploid peanut.