Name: Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9 (Video) | JoVE Journal
Uploaded: 2025-03-21T13:40:00.000+00:00
Duration: 6 min 47 s
Description: Learn CRISPR-Cas9 genome editing in Aedes aegypti mosquitoes for creating knockout and knockin lines. This protocol enables controlled gene expression and advances vector control research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors thank Judy Ishikawa and Ava Stevenson for helping with mosquito husbandry. This work was supported by funding from NIH awards (R01AI151004, RO1AI148300, RO1AI175152) awarded to O.S.A. and K22AI166268 to N.H.R. Figures were created using&#160;BioRender.

<ol><li>Anzalone, A. V., Koblan, L. W., Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAnzalone%2C+AV+%22Nat+Biotechnol%22">Nat Biotechnol</a>. 38 (7), 824-844 (2020).</li>
<li>Garneau, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+CRISPR%2FCas+bacterial+immune+system+cleaves+bacteriophage+and+plasmid+DNA%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.</a> Nature. 468 (7320), 67-71 (2010).</li>
<li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li>
<li>Shen, H., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+double-strand+break+repairs+and+their+application+in+plant+DNA+integration%5BTitle%5D)+AND+%22Genes+%28Basel%29%22%5BJournal%5D)">DNA double-strand break repairs and their application in plant DNA integration.</a> Genes (Basel). 13 (2), 322(2022).</li>
<li>Vinauger, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+host+learning+in+Aedes+aegypti+mosquitoes%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Modulation of host learning in Aedes aegypti mosquitoes.</a> Curr Biol. 28 (3), 333-344.e8 (2018).</li>
<li>Li, M., Bui, M., Yang, T., Bowman, C. S., White, B. J., Akbari, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germline+Cas9+expression+yields+highly+efficient+genome+engineering+in+a+major+worldwide+disease+vector%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector.</a> Proc Natl Acad Sci U S A. 114 (49), E10540-E10549 (2017).</li>
<li>Chaverra-Rodriguez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+delivery+of+CRISPR-Cas9+ribonucleoprotein+into+arthropod+ovaries+for+heritable+germline+gene+editing%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing.</a> Nat Commun. 9 (1), 3008(2018).</li>
<li>Rouyar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgenic+line+for+characterizing+GABA-receptor+expression+to+study+the+neural+basis+of+olfaction+in+the+yellow-fever+mosquito%5BTitle%5D)+AND+%22Front+Physiol%22%5BJournal%5D)">Transgenic line for characterizing GABA-receptor expression to study the neural basis of olfaction in the yellow-fever mosquito.</a> Front Physiol. 15, 1381164(2024).</li>
<li>Ang, J. X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Considerations+for+homology-based+DNA+repair+in+mosquitoes%3A+Impact+of+sequence+heterology+and+donor+template+source%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source.</a> PLoS Genet. 18 (2), e1010060(2022).</li>
<li>Zhang, J. -P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+precise+knockin+with+a+double+cut+HDR+donor+after+CRISPR%2FCas9-mediated+double-stranded+DNA+cleavage%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.</a> Genome Biol. 18 (1), 35(2017).</li>
<li>Coutinho-Abreu, I. V., Akbari, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technological+advances+in+mosquito+olfaction+neurogenetics%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">Technological advances in mosquito olfaction neurogenetics.</a> Trends Genet. 39 (2), 154-166 (2023).</li>
<li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+sex+determination+to+suppress+mosquito+populations%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Targeting sex determination to suppress mosquito populations.</a> eLife. 12, RP90199(2024).</li>
<li>Zhan, Y., Alonso San Alberto, D., Rusch, C., Riffell, J. A., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elimination+of+vision-guided+target+attraction+in+Aedes+aegypti+using+CRISPR%5BTitle%5D)+AND+%22Current+Biol%22%5BJournal%5D)">Elimination of vision-guided target attraction in Aedes aegypti using CRISPR.</a> Current Biol. 31 (18), 4180-4187.e6 (2021).</li>
<li>Greppi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mosquito+heat+seeking+is+driven+by+an+ancestral+cooling+receptor%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Mosquito heat seeking is driven by an ancestral cooling receptor.</a> Science. 367 (6478), 681-684 (2020).</li>
<li>Potter, C. J., Tasic, B., Russler, E. V., Liang, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Q+system%3A+a+repressible+binary+system+for+transgene+expression%2C+lineage+tracing%2C+and+mosaic+analysis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis.</a> Cell. 141 (3), 536-548 (2010).</li>
<li>Riabinina, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+and+expanded+Q-system+reagents+for+genetic+manipulations%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Improved and expanded Q-system reagents for genetic manipulations.</a> Nat Methods. 12 (3), 5 p following 222 219-222 (2015).</li>
<li>Labun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CHOPCHOP+v3%3A+expanding+the+CRISPR+web+toolbox+beyond+genome+editing%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing.</a> Nucleic Acids Res. 47 (W1), W171-W174 (2019).</li>
<li>Concordet, J. -P., Haeussler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPOR%3A+intuitive+guide+selection+for+CRISPR%2FCas9+genome+editing+experiments+and+screens%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens.</a> Nucleic Acids Res. 46 (W1), W242-W245 (2018).</li>
<li>Williams, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+fitness+costs+in+transgenic+Aedes+aegypti+mosquitoes%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Quantifying fitness costs in transgenic Aedes aegypti mosquitoes.</a> J Vis Exp. , e65136(2023).</li>
<li>Bassett, A. R., Tibbit, C., Ponting, C. P., Liu, J. -L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+targeted+mutagenesis+of+Drosophila+with+the+CRISPR%2FCas9+system%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.</a> Cell Rep. 4 (1), 220-228 (2013).</li>
<li>Coutinho-Abreu, I. V., Zhu, K. Y., Ramalho-Ortigao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgenesis+and+paratransgenesis+to+control+insect-borne+diseases%3A+current+status+and+future+challenges%5BTitle%5D)+AND+%22Parasitol+Int%22%5BJournal%5D)">Transgenesis and paratransgenesis to control insect-borne diseases: current status and future challenges.</a> Parasitol Int. 59 (1), 1-8 (2010).</li>
<li>Christian, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+DNA+double-strand+breaks+with+TAL+effector+nucleases%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Targeting DNA double-strand breaks with TAL effector nucleases.</a> Genetics. 186 (2), 757-761 (2010).</li>
<li>Kim, Y. G., Cha, J., Chandrasegaran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hybrid+restriction+enzymes%3A+zinc+finger+fusions+to+Fok+I+cleavage+domain%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.</a> Proc Natl Acad Sci U S A. 93 (3), 1156-1160 (1996).</li>
<li>DeGennaro, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((orco+mutant+mosquitoes+lose+strong+preference+for+humans+and+are+not+repelled+by+volatile+DEET%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET.</a> Nature. 498 (7455), 487-491 (2013).</li>
<li>McMeniman, C. J., Corfas, R. A., Matthews, B. J., Ritchie, S. A., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multimodal+integration+of+carbon+dioxide+and+other+sensory+cues+drives+mosquito+attraction+to+humans%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans.</a> Cell. 156 (5), 1060-1071 (2014).</li>
<li>Lobo, N. F., Clayton, J. R., Fraser, M. J., Kafatos, F. C., Collins, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+efficiency+germ-line+transformation+of+mosquitoes%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">High efficiency germ-line transformation of mosquitoes.</a> Nat Protoc. 1 (3), 1312-1317 (2006).</li>
<li>Kistler, K. E., Vosshall, L. B., Matthews, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+engineering+with+CRISPR-Cas9+in+the+mosquito+Aedes+aegypti%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti.</a> Cell Rep. 11 (1), 51-60 (2015).</li>
<li>Handler, A. M., Harrell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((2nd+Transformation+of+the+Caribbean+fruit+fly%2C+Anastrephasuspensa%2C+with+a+piggyBac+vector+marked+with+polyubiquitin-regulated+GFP%5BTitle%5D)+AND+%22Insect+Biochem+Mol+Biol%22%5BJournal%5D)">2nd Transformation of the Caribbean fruit fly, Anastrephasuspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP.</a> Insect Biochem Mol Biol. 31 (2), 199-205 (2001).</li>
<li>Harrell, R. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHarrell%2C+RA+%222nd+Mosquito+embryo+microinjection+under+halocarbon+oil+or+in+aqueous+solution%22">2nd Mosquito embryo microinjection under halocarbon oil or in aqueous solution</a>. 2024 (7), Cold Spring Harb Protoc. (2024).</li>
<li>Sun, R., Raban, R., Akbari, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generating+mutant+strains+with+transgenic+Cas9%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Generating mutant strains with transgenic Cas9.</a> Cold Spring Harb Protoc. 2023 (9), 671-678 (2023).</li>
<li>Giraldo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+expanded+neurogenetic+toolkit+to+decode+olfaction+in+the+African+malaria+mosquito+Anopheles+gambiae%5BTitle%5D)+AND+%22Cell+Rep+Methods%22%5BJournal%5D)">An expanded neurogenetic toolkit to decode olfaction in the African malaria mosquito Anopheles gambiae.</a> Cell Rep Methods. 4 (2), 100714(2024).</li>
<li>Liu, G., Lin, Q., Jin, S., Gao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+CRISPR-Cas+toolbox+and+gene+editing+technologies%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">The CRISPR-Cas toolbox and gene editing technologies.</a> Mol Cell. 82 (2), 333-347 (2022).</li>
<li>Chu, V. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+the+efficiency+of+homology-directed+repair+for+CRISPR-Cas9-induced+precise+gene+editing+in+mammalian+cells%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.</a> Nat Biotechnol. 33 (5), 543-548 (2015).</li>
<li>Laursen, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Humidity+sensors+that+alert+mosquitoes+to+nearby+hosts+and+egg-laying+sites%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Humidity sensors that alert mosquitoes to nearby hosts and egg-laying sites.</a> Neuron. 111 (6), 874-887.e8 (2023).</li>
<li>Weng, S. -C., Antoshechkin, I., Marois, E., Akbari, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+sex+separation+by+exploiting+differential+alternative+splicing+of+a+dominant+marker+in+Aedes+aegypti%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Efficient sex separation by exploiting differential alternative splicing of a dominant marker in Aedes aegypti.</a> PLoS Genet. 19 (11), e1011065(2023).</li>
<li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+confinable+gene+drive+system+in+the+human+disease+vector%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Development of a confinable gene drive system in the human disease vector.</a> eLife. 9, e51701(2020).</li>
<li>Dalla Benetta, E., et al. Engineered Antiviral Sensor Targets Infected Mosquitoes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDalla+Benetta%2C+E+%22The+CRISPR+journal%22">The CRISPR journal</a>. 6 (6), 543-556 (2023).</li>
<li>Li, H. -H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C-Type+lectins+link+immunological+and+reproductive+processes+in+Aedes+aegypti%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">C-Type lectins link immunological and reproductive processes in Aedes aegypti.</a> iScience. 23 (9), 101486(2020).</li>
<li>van Oers, M. M., Vlak, J. M., Voorma, H. O., Thomas, A. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+3%27+untranslated+region+of+baculovirus+p10+mRNA+in+high-level+expression+of+foreign+genes%5BTitle%5D)+AND+%22J+Gen+Virol%22%5BJournal%5D)">Role of the 3' untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes.</a> J Gen Virol. 80 (Pt 8), 2253-2262 (1999).</li>
<li>Salem, T. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+SV40+polyA+on+gene+expression+of+baculovirus+expression+vector+systems%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">The influence of SV40 polyA on gene expression of baculovirus expression vector systems.</a> PloS One. 10 (12), e0145019(2015).</li>
</ol>

O.S.A. is a founder of Agragene, Inc. and Synvect, Inc. with an equity interest. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. The remaining authors declare no competing interests.

CRISPR-Cas technology has changed the landscape of genome editing by promoting target-specific changes in chromosomes1. Even though transposable elements were essential for the generation of the first transgenic mosquitoes, their insertion sites are somewhat random, and the expression of the cargo construct (promoter + gene) may not correspond to the expression profile of the actual gene due to a genome positional effect (i.e., insertion site), which usually leads to ectopic expression<sup class="...

Precise genome editing has become arguably easier, but possible, with the establishment of the CRISPR-Cas technologies of molecular scissors1. These technologies take advantage of a mechanism that the prokaryotic immune system uses to fight against phage infections2. Amongst these systems, clustered, regularly interspersed, short palindromic repeats (CRISPR) along with the Cas9 nuclease usually rely upon 20 base pair RNAs, the guide RNAs (gRNAs), with sequences homologous to the targeted DNA, which are followed by an NGG protospacer adjacent motif (PAM) sequence3. The gRNAs loaded onto the Cas9 guide these nucleases precisely to specific target sites in the genome, triggering double-strand DNA breaks3. 
DNA double-strand breaks induce the repair mechanisms to patch the double-helix4. Whereas any DNA repair is expected to be precise, there exist less accurate DNA repair mechanisms that can leave behind sequence scars and, in turn, loss-of-function mutations4. Among the error-prone DNA repair mechanisms, the Non-Homologous End-Joining (NHEJ) can cause frame-shift mutations, including small deletions, insertions, and nucleotide changes (SNPs), that can result in loss-of-function mutation. The Homology Directed Repair (HDR) mechanism, on the other hand, relies upon the homologous chromosome as a template to copy the exact sequence of the undamaged allele and make a perfect repair of the targeted DNA sequence4.
Based on this knowledge, the CRISPR-Cas9 technology has been developed to precisely edit genomes, arguably at any sequence containing a PAM site3. In mosquitoes, the CRISPR-Cas9 technology has been used to knock out a variety of genes, through embryonic microinjection of a mixture of Cas9 and gRNAs, taking advantage of the NHEJ repair mechanism5,6. Similar germline mutagenesis is obtained with the injection of gRNA + Cas9 mix into the hemolymph of adult female mosquitoes7. This technology was coined ReMOT control and relies upon a modified version of a Cas9 tagged with a peptide that is taken up by the ovaries through endocytosis during the process of egg development (vitellogenesis)7. Knocking in specific gene cassettes into a genome is only possible through embryonic microinjection of a mixture of gRNA and Cas9 (or a plasmid expressing those molecules) along with a plasmid encoding a desired DNA cassette8. Taking advantage of the HDR mechanism, the plasmid containing the DNA cassette of interest flanked by homologous sequences (500-1,000bp)9,10 upstream and downstream of the target site is used as a template to rewrite the double-strand break, copying also the DNA cassette into the target sequence9. 
The CRISPR-Cas9 technology has been used to knock out multiple genes primarily involved with the sensory systems in the mosquito Aedes aegypti11, but also genes associated with male fertility and female viability (PgSIT) for population control12. Knocking out target genes has also been accomplished by knocking in genes encoding fluorescent markers into the coding sequences of specific genes13,14. This strategy has the advantage of not only inducing frame-shift mutations but also allowing the use of fluorescent light to sort the individuals of the new knockout line13,14. The A. aegypti genome has also been edited with sequences of binary expression systems, such as the Q-system (QF-QUAS)11. Knocking in the gene encoding the QF transactivator downstream to a promoter of a specific gene assures defined spatiotemporal expression of the transactivator15,16. Once a QF-expressing mosquito line is crossed to another mosquito line containing the binding sites (QUAS) for QF, the latter binds to it and triggers the expression of genes downstream to the QUAS sequence15,16. This system, overall, allows tissue- and time-specific expression of such effector genes, which can be fluorescent markers used for cell localization or detection of neuronal activity, and even Cas9 nucleases for disrupting genes in specific tissues (i.e., somatic knockout)11. 
Given all the information available for A. aegypti genetic transformation, we provide herein a detailed protocol with step-by-step directions to performing genome editing with the CRISPR-Cas9 system through embryonic microinjection. Strategies for generating both knockout, through frame-shift mutations and deletions mediated by NHEJ, and knockin lines, by HDR-mediated gene cassette insertions, are discussed.

<table><tbody><tr><td>10x Cas9 reaction buffer</td><td>PNA Bio&amp;nbsp;</td><td>CB01</td><td></td></tr><tr><td>Benchling software</td><td>Benchling</td><td>N/A</td><td>www.benchling.com</td></tr><tr><td>Cas9 dilution buffer</td><td>PNA Bio&amp;nbsp;</td><td>CB03</td><td></td></tr><tr><td>Cas9 protein</td><td>PNA Bio&amp;nbsp;</td><td>CP01-50</td><td></td></tr><tr><td>DH5&amp;alpha; E. coli Competent Cells</td><td>New England Biolabs</td><td>C2987</td><td></td></tr><tr><td>Double-sided sticky tape</td><td>Scotch Permanent</td><td>3136</td><td></td></tr><tr><td>Drosophila vials</td><td>Genesee Scientific&amp;nbsp;</td><td>32-109</td><td></td></tr><tr><td>Filter papers&amp;nbsp;</td><td>GE Healthcare Life Science&amp;nbsp;</td><td>1450-042</td><td></td></tr><tr><td>Fish food&amp;nbsp;</td><td>Tetra</td><td>B00025Z6YI</td><td>goldfish flakes&amp;nbsp;</td></tr><tr><td>Flugs</td><td>Genesee Scientific&amp;nbsp;</td><td>AS273</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Leica Microsystems&amp;nbsp;</td><td>&amp;nbsp;M165 FC</td><td></td></tr><tr><td>Gene fragment</td><td>Integrated DNA Technologies</td><td>N/A</td><td></td></tr><tr><td>gRNA</td><td>Synthego</td><td>N/A</td><td></td></tr><tr><td>Halocarbon oil 700&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H8898</td><td></td></tr><tr><td>Injection microscope</td><td>Leica Microsystems&amp;nbsp;</td><td>DM2000</td><td></td></tr><tr><td>JM109 &amp;nbsp;E. coli Competent Cells&amp;nbsp;</td><td>Zymo Research</td><td>T3005</td><td></td></tr><tr><td>Microinjector</td><td>Eppendorf</td><td></td><td>FemtoJet 4x&amp;nbsp;</td></tr><tr><td>Microloader Tips for Filling Femtotips&amp;nbsp;</td><td>Eppendorf</td><td>E5242956003</td><td></td></tr><tr><td>Micromanipulator&amp;nbsp;</td><td>Eppendorf</td><td></td><td>TransferMan 4r&amp;nbsp;</td></tr><tr><td>Micropipette Pullers&amp;nbsp;</td><td>Sutter Instrument&amp;nbsp;</td><td>P-2000</td><td></td></tr><tr><td>Microscope Cover Glass&amp;nbsp;</td><td>Fisherbrand&amp;nbsp;</td><td>12-542-B</td><td></td></tr><tr><td>Microscope slide&amp;nbsp;</td><td>Eisco</td><td>12-550-A3</td><td></td></tr><tr><td>Mouse blood (live mice used for feeding)</td><td>University of California</td><td>IACUC, Animal Use Protocol #S17187</td><td>Used for mosquito blood feeding; details comply with animal ethics protocols</td></tr><tr><td>NEB Q5 High-Fidelity DNA polymerase&amp;nbsp;</td><td>New England Biolabs</td><td>M0491S</td><td></td></tr><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28004</td><td></td></tr><tr><td>Plasmid Miniprep Kit&amp;nbsp;</td><td>Zymo Research</td><td>D4036</td><td></td></tr><tr><td>Quartz filament&amp;nbsp;</td><td>Sutter Instruments</td><td>QF100-70-10</td><td></td></tr><tr><td>Transcription Clean-Up Kit</td><td>Fisher Scientific</td><td>AM1908</td><td></td></tr><tr><td>Ultra-pure water&amp;nbsp;</td><td>Life Technologies</td><td>10977-023</td><td></td></tr></tbody></table>

genome editing in the yellow fever mosquito aedes aegypti using crispr-cas9

Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.

Design and validation of gRNA-mediated gene targeting for HDR homology recombination 
To ensure the desired gene is accurately targeted, we recommend selecting a couple of gRNAs and positioning the 5' and 3' homology arms close to the cutting site for HDR-mediated homologous recombination (Figure 1A). For example, we designed two gRNAs to target both sides of the start codon of the gene of interest and used the QF2-Hsp70-OpIE2-ECFP-SV40 ca...

Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering ...

genome-editing-yellow-fever-mosquito-aedes-aegypti-using-crispr

Research

JoVE Journal

Genetics

In this video, we demonstrate the delivery of gene drive construct into the embryo of Anopheles gambiae mosquito through embryo microinjection technique. This procedure allows for integration of  delivered DNA construct, through homologous recombination, into the germline cells to generate transformed mosquito population.

Mosquito Embryo Microinjection: A Technique to Deliver Exogenous DNA into Embryo Yolk of Anopheles Gambiae to Generate Germline Mutations

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Presented here is a protocol of Helicoverpa armigera (Hübner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the φC31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

Learn CRISPR-Cas9 genome editing in Aedes aegypti mosquitoes for creating knockout and knockin lines. This protocol enables controlled gene expression and advances vector control research.

Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

Explore More Videos