Figure 3. Generation of a large-scale sgRNA library.
(A) Design of the sgRNA pairs used for the HD_CFD library. sgRNAs were designed through CLD and filtered to target common exons in the 5’ORF and not overlap the start codon. sgRNAs were then paired to target two independent positions in the same gene. As an example the locations of the two target sites in ovo targeted by the two sgRNAs encoded in line HD_CFD000172 is shown. Exons are represented as boxes and regions in blue are protein coding. (B) Experimental strategy for the generation of the transgenic sgRNA library. sgRNA target sequences are encoded on oligonucleotides synthesized and cloned in pool. Individual plasmids are sequence verified and transformed into Drosophila at attP40 on the second chromosome following a pooled injection protocol followed by genotyping of individual transformants. (C) Applications of the pCFD6::FRT vector. pCFD6::FRT contains two non-compatible FRT sites either side of the sgRNA cassette. Using compatible FRT sides in trans allows to exchange sequences upstream or downstream of the sgRNAs in vivo. (D) Efficient promoter or sgRNA exchange in vivo. Summary of FLP/FRT mediated exchange of the sgRNA promoter (left) or sgRNAs (right). Each line represents a single sequenced animal. Red and blue boxes either side of the triangle (representing FRT) indicate successful recombination. (E) Summary statistics of the different functional groups present in the sgRNA library. Given is the number of genes from each category that are covered by fly lines, plasmids or against which currently no tools are available. Note that for some genes two fly lines or plasmids exist. Status in September 2019 is shown. Group ‘Others’ contains mainly genes with human orthologs associated with cancer development in humans.
Figure 3—figure supplement 1. Negative control sgRNA transgenes for use with HD_CFD library.
