CRISPR-mediated genome editing allows for efficient on demand creation of >200 kb deficiencies with precise boundaries.

Deficiency mapping remains a useful tool in the process of identifying causative genetic lesions in mutant strains isolated from forward genetic screens, in particular of non-coding mutants. However, there are significant areas across the genome with no deficiency coverage at all, and the boundaries of many deficiencies remain poorly defined. Here, we describe a simple methodology to generate balanced deficiency strains with up to 230 kb molecularly defined deletions (mini-deficiencies) using CRISPR/Cas9, thus providing a simple path for both precise and tailored deficiency mapping.