CAMIO for deletion analysis of endogenous DNA sequences in multicellular organisms

Interrogating the genome, especially locating critical cis-elements in non-protein coding regions, requires efficient deletion analysis. Conventional deletion analyses using synthetic constructs are cumbersome and nonphysiological. Here we invent CAMIO (Cas9-mediated Arrayed Mutagenesis of Individual Offspring), to achieve high-throughput organism-level structure-functional analysis of native DNA sequences. CAMIO permits creating arrays of targeted deletions directly in the genome. First, we assemble mutually exclusive gRNAs into a set. Combining two sets allows stochastic expression of many possible combinations of two gRNAs in germline stem cells. Second, we restrict Cas9 to male germ cells to elicit independent deletions around or between the gRNA-directed Cas9 cut sites in individual offspring. Thus, we can obtain from a single population cross a matrix of variable deletions covering a sizable DNA region at both coarse and fine levels. We exemplify the power of CAMIO in mapping the UTR sequences crucial for graded neuronal expression of chinmo.