CRISPR-Cas9 Genome Editing in Drosophila

Abstract

The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining (NHEJ) or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target-site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification, and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags.

Keywords: CRISPR; Cas9; Drosophila; genome engineering; homology directed repair.

Figure 1

Strategic planning flowchart. The options outlined in this protocol are indicated by green boxes. See text (Strategic Planning) for a detailed discussion of each choice point, including the advantages and disadvantages of each strategy.

Figure 2

gRNA plasmid cloning. The pU6-BbsI-gRNA vector contains two BbsI cut sites between the Drosophila U6-2 (snRNA:U6:96Ab) promoter and the common portion of the gRNA. Specific target site sequences are synthesized as complementary oligonucleotides designed to generate appropriate cohesive 5′ overhangs once annealed. The annealed oligonucleotides, once phosphorylated, are then ligated into the BbsI digested pU6-BbsI-gRNA vector. U6-2 sequence (blue), BbsI recognition sequences (red), target site specific sequence (green), and the common portion of the gRNA (grey) are indicated. Red arrowheads denote the breakpoints generated by BbsI cleavage.

Figure 3

Donor construct design. The (A) pHD-DsRed-attP vector and (B) pHD-DsRed donor vectors and their typical uses are depicted. Both vectors contain a removable 3xP3-dsRed marker flanked by LoxP sites and two multiple-cloning sites for insertion of the left (LHA) and right (RHA) homology arms. pHD-DsRed-attP also contains the recombination-based docking site attP. (A) In the case of replacing a locus using the pHD-DsRed-attP vector, two target sites flanking the region to be replaced are chosen. Homologous sequences immediately flanking the cleavage sites should be cloned into the MCSs. Upon Cas9-mediated cleavage and HDR, the region between the two gRNA cut sites is replaced with the attP site and removable DsRed marker. Using Cre recombinase, the DsRed marker can be removed leaving only the attP docking site and a single LoxP site. (B) In the case of tagging a gene using the pHD-DsRed vector, select a target site close to the tag insertion site and another target site in a nearby intron where the DsRed marker will be placed. Homology arms will include sequences immediately flanking the cleavage sites. In addition, one of the homology arms will contain the in-frame tag and sequences between the tag and the DsRed marker. Upon Cas9 cleavage and HDR, the untagged region is replaced with a tagged region and a visible 3xP3-DsRed marker. Using Cre recombinase, the DsRed marker can be removed leaving only the tagged coding sequence and a single LoxP site. Black arrows indicate the primer binding sites used for molecular characterization for candidate alleles. Note that the two locus specific primers are in the genomic region outside of the homology regions used in the donor vector.

Figure 4

Donor plasmid cloning. (A) Schematic of the pHD-DsRed-attP and pHD-DsRed donor vector including the MCSs. (B–D) Multiple cloning site sequences and primer design for type IIS restriction site (AarI or SapI) based cloning of homology arms for the LHA of pHD-DsRed-attP (B), the LHA of pHD-DsRed (C), and the RHA of both pHD-DsRed-attP and pHD-DsRed (D). Note that the LHA of pHD-DsRed-attP and pHD-DsRed requires slightly different primers due to the presence or absence of the attP site. Vector backbone sequences (grey), attP/LoxP sequence (purple), AarI/SapI recognition sites (red), and locus specific hybridization sequences (blue) are indicated. Red arrows indicate the breakpoints generated by AarI or SapI digestion.