Site-specific integrations at the Ae. aegypti kmo site were obtained by microinjection into preblastoderm embryos as previously described (57–59 (link)). For the kmoEGFP strain, the injection mix included 0.4 µg/µl of CRISPR/Cas9 enzyme (PNA Bio), 0.1 µg/µl of sgRNA-KmoEx4, and 0.3 µg/µl of donor plasmid pBR-KmoEx4 was microinjected to the Lvp wild-type embryos. The G2 kmoEGFP strain was utilized as a recipient for a second round of microinjections using sgRNA-HybRED, Cas9, and pSSA-KmoDR0.7 (same concentrations as above) to generate the kmoRG strain. Chromosomal integration of the transgenes at the kmo locus was confirmed by PCR analysis using genomic DNAs purified from a single G2 individual larva as the template and a primer set that is specific to the transgene or kmo (Fig. 1D ; Table S2, Supplementary Material ). PCR was performed using the Phusion High-Fidelity DNA polymerase (NEB) for 35 cycles: 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 2 minutes.
Crispr cas9 enzyme
Manufactured by PNA Bio
The CRISPR/Cas9 enzyme is a molecular tool used for gene editing. It consists of a guide RNA (gRNA) and a Cas9 enzyme. The gRNA binds to a specific DNA sequence, and the Cas9 enzyme then cleaves the DNA at that location, allowing for precise genetic modifications.
Automatically generated - may contain errors
3 protocols using crispr cas9 enzyme
1
Generation of Ae. aegypti kmo Transgenic Strains
2
Generating Kmo-Targeted Mosquitoes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A kmo-targeting plasmid donor, pBR-KmoEx4 (Chae et al., 2022 (link)), was injected to three embryo groups (~800 to ~ 1,200 per group) with various maternal levels of the NHEJ-associated gene (null/heterozygous/WT), descended from reciprocal parental crosses between the indel mutant (ku80−/−, lig4−/−, or DNA-PKcs−/−) and Lvp strains. The injection mix included 0.2 μg/μl of pBR-KmoEx4, 0.4 μg/μl of CRISPR/Cas9 enzyme (PNA Bio), and 0.1 μg/μl of sgRNA-KmoEx4 (Chae et al., 2022 (link)). Surviving G0 mosquitoes were outcrossed with the kmo−/− strain, which contains a TALEN-generated kmo-null mutant allele (Aryan et al., 2013a (link)), phenocopying the khw strain (Cornel et al., 1997 (link)). G1 larvae were screened for DNA repair event-dependent phenotypes of eye color and marker expression; white eye (Kmo-) is caused by end joining mechanisms, and the EGFP fluorescent body (Kmo-; EGFP+) is caused by the homology-directed repair mechanism.
3
CRISPR/Cas9-Mediated Gene Editing in Mosquitoes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For CRISPR/Cas9-based gene editing, Lvp embryos were microinjected using a solution containing 0.4 μg/μl of CRISPR/Cas9 enzyme (PNA Bio) and 0.1 μg/μl of sgRNAs (Supplementary Table S1 ); the latter of which were transcribed in vitro using MEGAscript T7 Transcription Kit (Ambion). The injection mix was incubated at 37°C for 20 min prior to microinjection to allow for the formation of CRISPR/Cas9-sgRNA complexes. Surviving G0 mosquitoes were outcrossed with Lvp, and individual G1 mosquitoes were examined for CRISPR-generated indel mutations by high-resolution melt analysis (HRMA) using the Phire Animal Tissue DirectPCR Kit (Thermo Scientific). The list of HRMA primers is presented in Supplementary Table S2 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!