Strategies for gene disruption in Drosophila

Abstract

Drosophila melanogaster has been a classic model organism for the studies of genetics. More than 15,000 Drosophila genes have been annotated since the entire genome was sequenced; however, many of them still lack functional characterization. Various gene-manipulating approaches in Drosophila have been developed for the function analysis of genes. Here, we summarize some representative strategies utilized for Drosophila gene targeting, from the unbiased ethyl methanesulfonate (EMS) mutagenesis and transposable element insertion, to insertional/replacement homologous recombination and site-specific nucleases such as the zinc-finger nuclease (ZFN), the transcription activator-like effector nuclease (TALEN) and the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. Specifically, we evaluate the pros and cons of each technique in a historical perspective. This review discuss important factors that should be taken into consideration for the selection of a strategy that best fits the specific needs of a gene knockout project.

Keywords: CRISPR; Cas9; Drosophila; Gene knock-out; Genome editing; Homologous recombination; Transposons.

Figure 1

The scheme of P-element transposition. The illustrations show a model of P-element mediated transposition. First, the transposase binds to sequences within both P-element termini and initiates a DSB at each end. The excised P-element could be translocated into a new target site to disrupt another gene (left). Gap repair can then generate duplicated target sites with the P-element sequences at each end. The 3′ extensions left at the donor site can be used for repair either from homologous sequences located in the other copy as a fully repaired gene which contains two adjacent P-element target site (top right), or by non-homologous end-joining for imprecise repair, which could generate products that contain varying lengths of P-element-derived sequences as the imprecisely repaired condition (bottom right).

Figure 2

The comparison of Ends-In and Ends-Out homologous recombination. Ends-In and Ends-Out are two paradigms for gene targeting. The major difference is whether the DSB is located within the region of homology (Ends-In) or at the ends (Ends-Out). The figure compares the basic outcomes of these two methods. With ends-in (left), a break is made within the region of homology. Recombination with the target results in a tandem duplication of all the homologous sequence carried on the donor, separated by any sequences that are between the FRT sites (in this case, the white ⁺ and I-CreI site). In contrast, Ends-Out provides a simple replacement event between the genome and the homologous sequence. The result is to interrupt the targeted gene with a modified, heterologous sequence, such as the w ⁺ marker (right).

Figure 3

Site-specific endonucleases. Three classic site-specific endonucleases including ZFN, TALEN and CRISPR/Cas9 are shown. (A) ZFN simply consists of a Zinc-finger protein (ZFP) fused to Fok1 endonuclease. The sequence composition of the α-helix in the zinc-finger determines the nucleotide binding specificity of the ZFP. As a result, a ZFP chain can be created by joining a few ZFPs together and generating high specificity, allowing Fok1 endonuclease to accurately cleave DNA at the target site. (B) The C-terminal end of a TALE contains a Fok1 endonuclease for DNA cleavage. The central part of the TALE contains a number of almost similar repeats that mediate specific binding to target loci in the genome, and each of these repeats specifically binds to one base of the target DNA via two amino acids named repeat variable di-residues (RVDs), including NG, NI, HD and HN (or NK) for recognizing one of the four different nucleotides: T, A, C and G, respectively [53, 54]. (C) Cas9 forms a sequence-specific endonuclease when complexed with the sgRNA. The Cas9/sgRNA complex then recognizes the targeted sequence, 20-bp in length, ending with two guanines (NGG) called the PAM site. Cleavage occurs on both strands upstream of the PAM sites. (D) The DSB is first induced by ZFN, TALEN or Cas9 endonuclease and then repaired by three possible mechanisms. When repaired by NHEJ, random deletions would occur at the site (left). When the repair is done by the endogenous template within the genome, the sequence would be fully repaired (middle). If an exogenous modified template is added, the sequence could be altered after repair, which is regarded as the gene editing (right).