Targeted genome engineering techniques in Drosophila

Abstract

For a century, Drosophila has been a favored organism for genetic research. However, the array of materials and methods available to the Drosophila worker has expanded dramatically in the last decade. The most common gene targeting tools, zinc finger nucleases, TALENs, and RNA-guided CRISPR/Cas9, have all been adapted for use in Drosophila, both for simple mutagenesis and for gene editing via homologous recombination. For each tool, there exist a number of web sites, design applications, and delivery methods. The successful application of any of these tools also requires an understanding of methods for detecting successful genome modifications. This article provides an overview of the available gene targeting tools and their application in Drosophila. In lieu of simply providing a protocol for gene targeting, we direct the researcher to resources that will allow access to the latest research in this rapidly evolving field.

Keywords: CRISPR; Drosophila; Gene targeting; Homologous recombination; NHEJ; Nuclease; TALEN; Zinc finger nuclease.

Figure 1

Diagrams of targetable nucleases. In the zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), each DNA-binding module is shown as a small, shaded oval; the FokI nuclease domains are large ovals. Each zinc finger contacts primarily 3 base pairs; each TALE module binds a single base pair. In both cases, two DNA-binding domains, each linked to one nuclease domain, are required to promote dimerization and cleavage. The CRISPR components are the Cas9 nuclease, a guide RNA that has about 20 bases of homology to the genomic target, and a small protospacer-adjacent motif (PAM) in the target.

Figure 2

Cellular mechanisms of double-strand break (DSB) repair. Sequence changes can be introduced following a nuclease-induced DSB both by the inaccurate nonhomologous end join (NHEJ) pathway and by homologous recombination (HR), using a donor molecule as a template. Shading in the HR product indicates that the frequency of donor sequence incorporation at the target decreases with distance from the break.

Figure 3

Paired nicking with CRISPRs. A modified Cas9 protein with one active site mutated (D10A) cuts only one strand of the target DNA. Providing two guide RNAs (gRNAs) directed to sequences in close proximity on opposite strands leads to an effective DSB.

Figure 4

A variety of donor designs have proven useful in templating HR. a. Long homologies in a circular plasmid donor allow introduction of large and small changes both close to and far from the nuclease-induced DSB. b. Single-stranded synthetic oligonucleotides can template small changes close to the break, including introduction of short sequence tags. c. Both ss oligos and long donors with homologies to sites flanking two DSBs can be used to delete large stretches of DNA, or to replace them with a desired sequence.

Figure 5

Detecting mutations with HRMA. a and b, HRMA of G0 flies. Each colored trace represents melting of PCR products from an individual fly. In a, a low level of mutation is detected in 3 of the flies tested (red and blue traces are displaced slightly from the rest). In b, most of the flies show high levels of mutation. c, HRMA of F1 flies produced by crossing G0’s to a balancer stock. Each trace represents a single fly derived from two of the G0’s shown in panel a.