One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

The purpose of this protocol is to generate an endogenous edited fly line. We show a fluorescent protein knock-in as an example, but it could also be applied to other knock-in, knockout, as well as mutation generation. Our protocol is a CRISPR homologous recombination based method, so there's nearly no restriction on mutation sites.

Moreover, our protocol greatly reduced the time and effort involved in genotyping steps. To begin, open the fly CRISPR website to design sgRNAs for Drosophila, and enter approximately 100 base pair sequences surrounding the desired knock-in location. Select CRISPR targets with the five prime guanine option.

Click the find CRISPR targets button to generate a list of possible sgRNA sequences. Then click the evaluate button for each sequence, and choose two sgRNAs that have zero off-targets. Since the natural protein expression levels are typically much lower, select fluorescent tags that are bright and photostable, and fit the experimental setup.

Next, design a linker to link epitope tags to the fluorescent marker for biochemical analysis. Use a linker peptide ranging from two to 20 amino acids, comprising amino acids like glycine and serine. To design the donor vector for Drosophila, organize the DNA sequence of the linker peptide and the fluorescent tag, and ensure homologous sequences flanking both ends.

Modify any PAM sequences within the donor plasmid to maintain the resulting proteins amino acid sequence. Next, position the start and stop codons depending on the tag location. For internal tagging, ensure the fluorescent tag is in frame with the coding sequence of the target gene.

For N-terminus tagging, position the fluorescent tag after the endogenous start codon. For C-terminus tagging, position the fluorescent tag before the natural stop codon. Ensure to retain only a single start codon to avoid potential translation issues.

Use the alternative restriction enzyme based cloning approach to insert the sgRNA sequence into the plasmid U6 Bbs1-chiRNA plasmid. After creating the sgRNA plasmid, transform the plasmid in DH5-Alpha Escherichia coli as per the manufacturer's instructions. Verify the resulting clones through Sanger sequencing, using either T3 or T7 standard sequencing primers.

To create the donor plasmid, use double-stranded DNA with the desired sequence, and integrate it into the plasmid Bluesript 2SK negative multiple cloning site. Sequence the donor plasmid to ensure sgRNA or PAM sequences are correctly modified and there are no SNPs present in the plasmid. Next, take Cas9 flies injected with the constructed gRNA and donor plasmids.

Anesthetize the flies with carbon dioxide, and separate male flies and virgin females. To establish fly crosses, pair each G zero male with at least two FM7L virgin females in individual narrow CT food vials. Place crossed vials in a vented incubator maintained at 25 degrees Celsius and 60%humidity until F1 flies eclose.

Then anesthetize and collect at least 10 virgin female offspring from both G zero male and female crosses. Ensure each F1 fly is tagged with the details of its parentage and stored separately. To screen flies for PCR, design primers with a melting temperature of around 55 degrees Celsius that encompass the editing site of the target gene.

Prepare lysis buffer for each fly sample, and dispense 10 microliters into each well of a 96 well PCR plate. Maintain the buffer at a cold temperature during the leg dissection process. Next, prepare glass capillary tubes, filling one end with sucrose agar food.

Place these tubes in a 96 well deep well plate, referred to as the fly hotel. For leg dissection, position the carbon dioxide anesthetization pad under a stereo microscope. Anesthetize a candidate fly, and sever one of its middle legs.

After that, immerse the leg into the lysis buffer. Hold the fly wing with a pair of fine metal forceps. Move the fly to the fly hotel, and seal the tube with yarn immediately.

House the fly hotel in a vented incubator maintained at 25 degrees Celsius and 60%humidity. Submerge the severed legs in the lysis buffer solution in a thermal cycler. Organize the PCR reaction as instructed by the manufacturer, using 1.5 microliters of the fly leg lysate as the DNA source.

Subject the PCR products to agarose gel electrophoresis, and interpret the results based on band size. Based on the gel results, select the positive flies from the fly hotel and individually cross them with balancer flies to produce the F2 generation. Use homozygous stocks for Sanger sequencing of the editing site to ensure accuracy.

Back-cross validated fly lines with wild type flies to remove background mutations. Screen each generation using the single leg PCR protocol. Confocal microscope images of adult Drosophila brains showed period protein organized into discreet nuclear foci throughout the late night and early morning time points.

Overall, this protocol is robust if your genotyping PCR primers are specific.