Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species

The ABCs of CRISPR in Tephritidae: developing methods for inducing heritable mutations in the genera Anastrepha, Bactrocera and Ceratitis

“…Although species development time and morphology resulted in variation in the methods with regards to time of injection and location of injection, adult survival rate for all species was about the same and ranged between 1.4% ( C. capitata , n = 927) and 2.3% ( B. dorsalis , n = 1176) (Table ). These survival rates are much lower than in other studies that have demonstrated germline transformations in Tephritidae following the microinjection of plasmids (6% in A. ludens and B. dorsalis and 11–39% in C. capitata ) (Handler and McCombs, ; Ogaugwu et al, ; Schetelig et al ., ; Eckermann et al ., ), and this is probably because of our aggressiveness in ensuring that all embryos received a dose of the Cas9 protein and sgRNA cocktail. Despite the similarity in survival rates that we achieved in our species, the number of visible mutants recovered varied widely and ranged between 0% ( B. dorsalis zero out of 27 surviving G0 adults) and 28% ( C. capitata , seven out of 25 surviving G0 adults).…”

Improvement and Use of CRISPR/Cas9 to Engineer a Sperm-marking Strain for the Invasive Fruit Pest Drosophila suzukii

“…To generate the embryonic driver line 06_F5M2 by random piggyBac integration, the transformation vector HMMA006 and the helper plasmid MK006 (58) were mixed at a final concentration of 400 and 200 ng/µl respectively. To validate that the transgene represents a single integration even, we performed inversePCR as described (58) For the transgene editing experiments using CRISPR/Cas9, DNA was mixed at a concentration of 400, 150, and 350 ng/µl for Cas9 (HMMA056), gRNA (HMMA102, HMMA103, or HMMA104), and donor plasmid HMMA097, respectively. Higher concentration was used at 400, 250, and 400 ng/µl, respectively.…”

Improvement and Use of CRISPR/Cas9 to Engineer a Sperm-marking Strain for the Invasive Fruit Pest Drosophila suzukii

“…All embryonic injections were performed using transfection grade plasmid preparations without further precipitation steps. To generate the embryonic driver line 06_F5M2 by random piggyBac integration, the transformation vector HMMA006 and the helper plasmid MK006 (58) were mixed at a final concentration of 400 and 200 ng/µl respectively. To validate that the transgene represents a single integration even, we performed inversePCR as described (58) using XhoI and EcoRI restriction enzymes.…”

“…To generate the embryonic driver line 06_F5M2 by random piggyBac integration, the transformation vector HMMA006 and the helper plasmid MK006 (58) were mixed at a final concentration of 400 and 200 ng/µl respectively. To validate that the transgene represents a single integration even, we performed inversePCR as described (58) using XhoI and EcoRI restriction enzymes. For both the 5 and 3' junctions, we each obtained only a single fragment, whose sequences For the transgene editing experiments using CRISPR/Cas9, DNA was mixed at a concentration of 400, 150, and 350 ng/µl for Cas9 (HMMA056), gRNA (HMMA102, HMMA103, or HMMA104), and donor plasmid HMMA097, respectively.…”

“…Injection needles were prepared as previously described (58) .To inject in D.suzukii embryos, the eggs have to be squeezed out of the apple agar plates individually using home-made closed-tip glass pipettes. Embryos were then de-chorionated for 3 minutes using generic Clorox (DanKlorix, CP GABA GmbH, Hamburg, Germany) containing 2.5% sodium hypochlorite at final concentration of 1.25% sodium hypochlorite and washed in washing buffer (100mM NaCl, 0.02% Triton X-100) followed by thorough wash with desalted water.…”