Continual improvement of CRISPR‐induced multiplex mutagenesis in Arabidopsis

SUMMARY CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of the promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side by side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported. We genotyped more than 6000 Arabidopsis T2 plants to test seven promoters and six types of NLSs across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We found that the RPS5A promoter and bipartite NLS were individually the most efficient components. When combined, 99% of T2 plants contained at least one knockout (KO) mutation and 84% contained 4‐ to 7‐plex KOs, the highest multiplexing KO rate in Arabidopsis to date. These optimizations will be useful to generate higher‐order KOs in the germline of Arabidopsis and will likely be applicable to other CRISPR systems as well. Significance Statement Efficient CRISPR systems are essential for large‐scale experiments. We systematically compared different promoters and nuclear localization sequences side by side for multiplex mutagenesis in Arabidopsis using phenotyping and multiplex amplicon sequencing of thousands of plants. The combination of the RPS5A promoter with the double‐BP NLS results in essentially all plants containing at least one knockout (KO) mutation and the majority with at least four KO mutations.