Drosophila Seamless Gene Editing

Genome editing techniques can artificially alter the genetic information of Drosophila. However, there is an increasing demand for precise mutation manipulation. Especially, in Drosophila genetic manipulation, various selection markers are usually introduced to improve the screening efficiency of mutants. This creates a conflict between screening efficiency and the introduction of unrelated sequences. Not only markers, but also recombination residuals. These additional sequences may interfere with the normal function of regulatory sequences located in the 5' region of the target gene or adjacent genes. This is a fairly common problem in Drosophila, as about 31.6% of genes are located in head-to-head orientation, and the promoters of these genes are often closely spaced, with transcription start sites (TSS) less than 400bp apart.

Therefore, it is important to establish a seamless gene editing system for accurate site-specific gene editing. We aim to select the optimal seamless editing strategy for our clients to construct footprint-free and accurate fruit fly mutants for specific research objectives and gene characteristics.

Fig.1 Drosophila Seamless Gene Editing Strategies

We offer several CRISPR seamless editing schemes according to different experimental purposes and genetic characteristics: CRISPR/Cas9 combined with piggyBac strategy and two-step homologous recombination strategy.

• CRISPR/Cas9 combined with piggyBac strategy 

  CRISPR/Cas9 is used to introduce mutations and screening marker, and then the piggyBac transposase system precisely excises irrelevant sequences. The piggyBac transposon system recognizes specific inverted terminal repeat sequences (ITRs) at both ends of the DNA and inserts it into the TTAA locus in the chromosome. This strategy is simple and effective and does not require secondary genetic manipulation.

• sgRNA Plasmid Constructs
• Donor DNA Constructs

  piggyBac needs TTAA site. We provide donor DNA design synthesis services, including mutant gene codon optimization, selection of selectable markers (e.g., DsRed, mini-white, vermillion), design of 1~2 kb 5' and 3' homology arm and TTAA sequences at both ends, as well as optimizing the distance between TTAA sequence and the target mutation site.

• Acquisition of Gene Knock-in Drosophila Mutants

  Drosophila embryos with Cas9 gene are microinjected, and healthy females and males are crossed with w¹¹¹⁸ respectively, and the progeny carrying dominant markers are screened. Then balancing is required. We will confirm the mutants by sequencing.

• Excision of Screening Marker Gene

  We offer a series of endogenously stably expressed piggyBac strains that eliminate unrelated sequences by simple hybridization alone. The correctly identified transgenic Drosophila strain is crossed with those. Individuals from the progeny that do not carry the dominant marker are selected, and the cross is continued to select homozygous. The genomic DNA of the homozygous is extracted, and PCR amplification and sequencing are performed near the mutation site to determine whether the homozygous strains carried target mutation without irrelevant sequence.

• Two-step homologous recombination strategy 

  Theoretically, this approach allows for a seamless insertion of any gene at any locus in the Drosophila genome. And it can be used in many transgenic systems, such as MiMIC and RMCE. The first homologous recombination replaces the target gene with a marker gene, and the second homologous recombination replaces the marker gene with the target gene with a mutation. In this case, both ends of the target gene can carry different sgRNA sequences than those used for genomic shearing to ensure that Cas9 does not act on the homologous arm vector.

• sgRNA Plasmid Constructs
• Donor DNA Constructs

  The point of the two-step homologous recombination is two mutually exclusive sets of sgRNA and donor DNA. Usually, we alter the nucleotide sequence at both ends of the selectable gene by first recombination, thus preventing cleavage of the donor construct or the modified DNA. Besides, the primary marker used for screening requires to be compact and easy to insert by HDR.

• Acquisition of Gene Knock-in Drosophila mutants

  Donor templates are injected into germline-specific Cas9 lines embryos along with sgRNA expression plasmids, and then screen healthy progeny with dominant marker, where insertion of the marker can simultaneously generate strong null allele mutants. Similarly, gene-level analysis methods are used to identify the true mutants.

• Excision of Screening Marker Gene

  The insertion of donor DNA we designed generates 2 new Cas9 target sites that are not present in the endogenous sequence and can be used to remove the inserted cassette by a second step of HDR for final replacement. Our service generally provides homozygous strains and if there is a homozygous death, then balancing is necessary and we are supposed to state that in our final report.

• We follow up with you regularly during the project for progress and next strategies to ensure the project is completed on time. If any problems or complications occur, we will contact you to provide a timely solution. Once completed, we provide clients with a detailed report including strategy design, results and raw data.

High specificity - Complete sequence and prediction platform ensures that the sgRNA can smoothly match with the corresponding sequence in the genome and correctly guide Cas9 to cleave the target sequence. sgRNAs for Drosophila in vivo may be confirmed first in vitro (optional).

Various genome seamless editing services - Point mutations in exon and in intron, domain deletion, gene replacement, and so on. Besides, a broad technology platform allows us to offer a combination of seamless genetic transformation strategies, including CRISPR/Cas9, RMCE and MiMIC.

Fast turnaround - Experienced microinjection and molecular design assembly platforms ensure that seamless mutant flies are obtained in 4~6 weeks without other services.

Success rate - A one-on-one customization project helps clients choose the most suitable seamless editing solution with guaranteed success rate and high quality.