Figure 1. A sequential CRISPR scheme for targeted editing of the Rsp satellite locus.

A) We implemented a CRISPR scheme to make sequential edits to repetitive loci and demonstrate its utility with the Rsp satellite—a complex satDNA in the pericentric heterochromatin of 2R in D. melanogaster. The scheme involves a set of crosses (Figs S2, S4) to establish stable fly lines with altered Rsp loci (panel B) and no changes in flanking sequences. We indicate target site locations for two guide RNAs with complementary distributions along the Rsp locus—Rsp-g1 (blue down arrows) and Rsp-g2 (green up arrows)—of the Iso1 parent allele, a subline with a putative deletion (Iso1ΔC16) following CRISPR with Rsp-g1, and a subline with a large deletion (Iso1ΔC16Δ20) following sequential CRISPR with Rsp-g2. Both gRNAs target sites specifically matching Rsp repeat variants (blue) and avoid non-Rsp sequences in the locus (grey). Editing with this scheme occurs at high efficiency: few guide target sites remain following CRISPR (3/31 Rsp-g1 in Iso1ΔC16; 1/99 Rsp-g2 in Iso1ΔC16Δ20). B. We obtain a similar distribution of Rsp allele sizes after the first round of CRISPR editing, regardless of the method—injection or crossing (with two independent Rsp-g1 experiments—cross 1 and cross 2; see Fig S2). The sequential CRISPR cross shifted the resulting allele distribution towards smaller sizes.