Fig. 1.

Experimental overview. Injection mixtures containing pHsp70-Cas9, the appropriate pBS-U6-gRNA(s), and, for gene replacement, an ssDNA donor are injected into pre-blastoderm embryos. Twenty-four hours after injection, a subset of embryos can be assayed molecularly for the presence of targeted modifications to assess efficiency. Rear remaining embryos to adulthood and outcross to recover heritable modifications. Methods for identifying flies with the targeted modification include (1) phenotypic screening if the phenotype of the targeted modifications is known and readily observable, (2) molecular screening by PCR-based analysis, (3) negative screening for the removal of a marked transposable elements in the targeted locus, and (4) positive screening using a dsDNA donor with a visible marker