Precise and adaptable knock-out generation in wheat thanks to a multi-stop cassette

The advent of clustered regularly interspaced short palindromic repeat (CRISPR) genome editing initiated an era of accelerated understanding of gene function. A key approach to achieve this goal is functional validation, notably by knock-out (KO) generation of candidate genes in a genetic background harboring the character of interest. CRISPR genome editing enables precise genetic targeting, but the generated mutations are random. In this study, we propose a simple and straightforward method for precise KO (PKO) generation, using CRISPR-Cas9 for precise targeting coupled with a 32bp multi-stop cassette. This cassette is harboring a STOP codon in every reading-frame, such as its insertion guarantees the presence of a stop codon at the position of insertion. The CRISPR-Cas9 system and multi-stop cassette was bombarded on wheat embryos using biolistic. From the 391 embryos bombarded, 177 events of insertion were detected. Sanger sequencing and Next Generation Sequencing (NGS) showed that, in the majority of events, the cassette was truncated prior to its insertion, on average by 5bp. By design, this means that in the majority of detected events a multi-stop sequence was successfully inserted at the targeted position. Only one perfect insertion event was observed and the number of cells reached by the cassette is low, which was expected from this design, indicating that improvements can be made to the method. Our findings showed that PKO is achievable in wheat calli, which opens the way to improvements on wheat, and future developments of optimized KO on regenerated wheat plants.