Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9

Abstract

The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.

Keywords: HRMA; nanos-Cas9.

Fig. 1.

Optimization of the Drosophila Cas9/sgRNA system. (A) The nos-Cas9 plasmid encodes the S. pyogenes Cas9 gene under the regulation of the nos promoter 5′ UTR and 3′ UTR. The attB sequence and a vermillion⁺ marker are also included in this plasmid. This plasmid was used either directly for mutagenesis in microinjection experiments or for insertion into attP sites to generate Cas9 transgenic flies. (B and C) Schematics of the sgRNA constructs used in this study. (B) Three versions of plasmids expressing sgRNA (U6a, U6b, or minimal nos promoter). (C) Two sgRNA scaffold versions, one similar to that described by Mali et al. (10) and the other with 10 extra base pairs inserted between the crRNA and the tracrRNA modules. The extra base pairs are underscored.

Fig. 2.

Testing the optimized Drosophila nos-Cas9/sgRNA system on the white locus. (A) Schematic of two sgRNAs targeting the white locus, with the 20-nt target sequence underscored and PAM in bold type. Gray boxes represent exons. w1 sgRNA targets the first exon, and w2 targets the second. (B) RT-PCR results confirming the expression of Cas9 in {nos-Cas9}attP40 and {nos-Cas9}attP2 embryos, with actin 5C as an internal control. (C and D) Representative sequencing results showing the indel mutations generated in this study, with w1 sgRNA (C) and w2 sgRNA (D). (E) Schematic showing the defined deletion generated by using w1 and w2 sgRNAs, along with representative sequencing results showing the break points of the deletions generated. The genomic sequence between the sgRNA target sites is not shown. In C–E, the targeted genomic DNA sequence is underscored, and the NGG PAM sequence is in bold type. Dashed lines represent the locations of genomic deletions detected by sequencing.

Fig. 3.

No off-targets were detected using the optimized Drosophila Cas9/sgRNA system to generate heritable mutations. (A and B) Potential off-target sites for w1 sgRNA (A) and w2 sgRNA (B). Genomic regions that have at least 11-nt homology to the sgRNA seed region and a neighboring PAM sequence were sequenced from F1 white-eyed flies generated by the optimized system. Mismatches between the potential off-targets and the targeted region are underscored. The PAM sequences are in bold type.