The overall goal of this method is to generate a highly tissue-specific targeted expression system. In this method, we demonstrated the generation of a binary transcriptional driver specific for our Drosophila FGF homolog, branchless. Dynamic, spatiotemporal expression of branchless regulates branch hemogenesis of tracheal airway epithelium.
Though this method can provide insight into the spatiotemporal expression and migration of branchless producing cells, it can be easily be applied to other genes and tissues in Drosophila or in other model organisms, such as C.Elegans and zebrafish. The main advantage of this technique is that it generates expression systems that are highly reliable, tissue and gene specific, and active exclusively in cells where the cis regulatory elements of the replaced gene are functional. Binary transcription systems are essential tools for targeted gene expression and manipulation.
A trans activator, such as GAL4 or LexA, expressed under the control of cis regulatory elements of a gene, drive an effector trans gene that's placed downstream of specific transcription binding sites, like UAS for GAL4 and/or LexO for LexA. This methodology replaces the first coding exon of the branchless gene with the transcription driver. The CRISPR Cas9-based method places a LexA trans activator sequence under the endogenous regulation of the branchless gene, and consequently, facilitates trans activator expression exclusively in branchless specific spatiotemporal patterns.
Begin by selecting two guide RNA target sites that specifically target two ends of the selected region of interest. Open the CRISPR design tool of choice. Fly CRISPR Optimal Target Finder is used here.
Click on Select Genome'insert the sequence of interest, and select the most recent uploaded Drosophila melanogaster genome annotation, currently R underscore six, in the drop-down menu. Next, click Select guide length'and input 20. Select All CRISPR targets'and click Find CRISPR targets.
Evaluate all candidate guide RNA targets by setting maximum for stringency and NGG only'for PAM. To generate a tandem guide RNA expression vector, first PCR amplify a DNA fragment to introduce two proto spacer sequences into a pCFD4 RNA expression vector backbone. Use a forward and reverse primer, each with a proto spacer sequence, and overlap with the pCFD4 vector sequence in a PCR using high-fidelity polymerase and pCFD4 template DNA.
To prepare pCFD4 for cloning, digest the plasmid with Bbs1 enzyme. Set up a reaction in the appropriate digestion buffer containing two to five micrograms of pCFD4 plasmid and one microliter of Bbs1 enzyme and a final volume of 50 microliters. Mix the reaction contents by gentling tapping the tube and collecting all the scattered droplets from the wall of the tube to the bottom by a brief spin.
Incubate the reaction mix at 37 degrees Celsius for two hours after running the PCR product and digested plasmid on a one percent agarose gel. Purify the 6.4 kilobase pair linear plasmid and 600 base pair PCR product using standard gel purification columns. Set up the DNA assembly reaction as per the manufacturer's protocol.
For genomic knock-in of a GAL4 or LexA sequence, design a double-stranded HDR donor containing the trans activator sequence, flanked by two homology arms. To avoid re-targeting of the guide RNA to the engineered locus, design the replacement donor in such a way that the guide RNA recognition sites are disrupted by the exogenous sequence introduced. Also, to avoid altering the purity of cis regulatory elements, always select the guide RNA recognition sites within the coding exon region.
To generate a replacement cassette, plan the DNA assembly strategy to join four segments together. The five prime and three prime homology arms, the middle exogenous trans activator DNA sequence, and the linearized cloning vector backbone. PCR amplify a GAL4 or a LexA expression cassette from an appropriate vector and PCR amplify the homology arms from the genomic DNA extracted from the parent fly line selected for injection.
Use high-fidelity polymerase in each PCR to generate the target fragments. To set up the DNA assembly reaction, mix the linearized cloning vector and target fragments generated by PCR cloning with the DNA assembly mastermix and a final reaction volume to 20 microliters as per the manufacturer's instructions. Incubate the reaction mix for one hour at 50 degrees Celsius.
When Nos-Cas9 embryos previously injected with both the guide RNA expression plasmid and the replacement donor, develop into adults, cross G0 flies individually to balance your flies from a line appropriate for the targeted allele. Anesthetize the F1 offspring from each G0 cross on a carbon dioxide pad. Randomly pick 10 to 20 males under a stereo microscope.
Individually cross each selected male to a balancer female from a line appropriate for the targeted allele. When the F2 larvae hatch, pick the F1 father from each cross. Extract genomic DNA by squishing each fly for 10 to 15 seconds with a pipette tip, containing 50 microliters of squishing buffer without dispensing the buffer.
Then, dispense the remaining buffer into the tube and mix well. Incubate at 37 degrees Celsius for 20 to 30 minutes. Following the incubation, place the tubes in 95 degree Celsius heat block for one to two minutes to inactivate the proteinase K.After spinning down for five minutes at 10, 000 times G, store the preparation at four degrees Celsius for further PCR analysis.
Perform three-step PCR-based screens using primers forward one and reverse one to screen for the existence of the insertion or replacement. Perform PCR using forward two and reverse two primers to verify the insertion or replacement from the three prime region. Perform PCR using primers M13F and reverse three to check ends-in'HDR.
Keep the fly lines with the confirmed ends-out'HDR and establish balanced stocks from the F2 generation. Out-cross the balancer flies again to remove any unintended mutations on other chromosomes. Branchless, or bnl expression, is shown here in red in the third instar larvae wing disc, with the receptor breathless expressing cells, shown in green, in the growing air sac primordium.
Branchless expressing cells are shown here in the TR5 transverse connective branch and TR2 dorsal branch. Breathless expressing tracheal cells are shown here in green. Breathless expressing cells are shown in green, marking the developing embryonic trachea from stage nine to stage 14 and the branchless expressing cells are shown in red.
This image shows branchless expression induced in ectopic tissue locations under hypoxic conditions, relative to control. This method was designed to retain all the original spatiotemporal regulations of branchless gene transcription. Therefore, it was important to replace only the first coding exon of the gene with the LexA trans activator coding sequence.
After its development, this new genetic toolset paved the way to explore novel tissue expression patterns of the branchless gene. It also enabled gene mis-expression and knocked down exclusively in branchless expressing cells and helped in monitoring linear migration and signaling interactions of the cells.
