Cas9 Targeting and Specificity in Higher-Order CRISPR Multiplexed Plants

Many gene families are highly redundant and have diverse chromosomal locations in plants necessitating higher order mutant construction. CRISPR-Cas9 has emerged as a powerful tool to create targeted mutations in multiple loci simultaneously. The effective targeting of these gene families necessitates high specificity for the target without affecting other genomic loci. Previous work based on small scale targeting has suggested that the off-target rate for Cas9 in Arabidopsis is low or absent. Experiments involving higher order multiplex targeting have increased potential for off-target effects, which has implications in the confirmation of mutants. To clarify these issues, we have performed whole genome resequencing of Arabidopsis plants targeting 14 genomic loci at once. We demonstrate that Cas9 is highly specific in Arabidopsis with no detectable off-target effects. Our work also demonstrates that chromosomal translocation events are extremely rare. As such, the wild type Streptococcus pyrogenes Cas9 is specific enough to make off-targeting events undetectable in Arabidopsis. We have used this system to target several large gene families and will present data on the mutational analysis of multi-gene families regulating plant development.