The expansion of gene families via duplication is a characteristic feature of plant genomes and presents a major challenge for assigning biological functions using single-gene knockouts. Here, we present a strategy for analyzing the biological functions of highly expanded enzyme families in A. thaliana, using the cytochrome P450 subfamily 71B as an example. This subfamily contains 32 expressed genes, and only a few are functionally characterized, such as CYP71B15 (PAD3), an essential component of a camalexin biosynthetic complex. Aiming for generation of a complete 32x knockout, we have deleted four CYP71B clusters (up to 83 kb in size) using CRISPR/Cas9-based gene editing. Metabolite compositions are being systematically analyzed via UHPLC/Q-TOF-MS suggesting functions contributing to defense and/or communication. Here, a cyp71b11-cyp71b13 deletion mutant is impaired in the formation of hydroxycamalexin glycosides, which results in enhanced camalexin autotoxicity.