Name: genome editing in the yellow fever mosquito aedes aegypti using crispr-cas9
Uploaded: 2025-03-21T13:40:00.000+00:00
Duration: 6 min 47 s
Description: The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering ...

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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+with+CRISPR-Cas+nucleases,+base+editors,+transposases+and+prime+editors.">Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.</a> Nat Biotechnol. 38 (7), 824-844 (2020).</li><li>Garneau, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR/Cas+bacterial+immune+system+cleaves+bacteriophage+and+plasmid+DNA.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double-strand+break+repairs+and+their+application+in+plant+DNA+integration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+host+learning+in+Aedes+aegypti+mosquitoes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+Cas9+expression+yields+highly+efficient+genome+engineering+in+a+major+worldwide+disease+vector.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+CRISPR-Cas9+ribonucleoprotein+into+arthropod+ovaries+for+heritable+germline+gene+editing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+line+for+characterizing+GABA-receptor+expression+to+study+the+neural+basis+of+olfaction+in+the+yellow-fever+mosquito.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+homology-based+DNA+repair+in+mosquitoes:+Impact+of+sequence+heterology+and+donor+template+source.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+precise+knockin+with+a+double+cut+HDR+donor+after+CRISPR/Cas9-mediated+double-stranded+DNA+cleavage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technological+advances+in+mosquito+olfaction+neurogenetics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+sex+determination+to+suppress+mosquito+populations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+vision-guided+target+attraction+in+Aedes+aegypti+using+CRISPR.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+heat+seeking+is+driven+by+an+ancestral+cooling+receptor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Q+system:+a+repressible+binary+system+for+transgene+expression,+lineage+tracing,+and+mosaic+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+and+expanded+Q-system+reagents+for+genetic+manipulations.">Improved and expanded Q-system reagents for genetic manipulations.</a> Nat Methods. 12 (3), 219-222 (2015).</li><li>Labun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHOPCHOP+v3:+expanding+the+CRISPR+web+toolbox+beyond+genome+editing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPOR:+intuitive+guide+selection+for+CRISPR/Cas9+genome+editing+experiments+and+screens.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+fitness+costs+in+transgenic+Aedes+aegypti+mosquitoes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+targeted+mutagenesis+of+Drosophila+with+the+CRISPR/Cas9+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+and+paratransgenesis+to+control+insect-borne+diseases:+current+status+and+future+challenges.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+double-strand+breaks+with+TAL+effector+nucleases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+restriction+enzymes:+zinc+finger+fusions+to+Fok+I+cleavage+domain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=orco+mutant+mosquitoes+lose+strong+preference+for+humans+and+are+not+repelled+by+volatile+DEET.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+integration+of+carbon+dioxide+and+other+sensory+cues+drives+mosquito+attraction+to+humans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+germ-line+transformation+of+mosquitoes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+with+CRISPR-Cas9+in+the+mosquito+Aedes+aegypti.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2nd+Transformation+of+the+Caribbean+fruit+fly,+Anastrephasuspensa,+with+a+piggyBac+vector+marked+with+polyubiquitin-regulated+GFP.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2nd Mosquito embryo microinjection under halocarbon oil or in aqueous solution. 2024 (7), (2024).</li><li>Sun, R., Raban, R., Akbari, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+mutant+strains+with+transgenic+Cas9.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+neurogenetic+toolkit+to+decode+olfaction+in+the+African+malaria+mosquito+Anopheles+gambiae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR-Cas+toolbox+and+gene+editing+technologies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+efficiency+of+homology-directed+repair+for+CRISPR-Cas9-induced+precise+gene+editing+in+mammalian+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humidity+sensors+that+alert+mosquitoes+to+nearby+hosts+and+egg-laying+sites.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+sex+separation+by+exploiting+differential+alternative+splicing+of+a+dominant+marker+in+Aedes+aegypti.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+confinable+gene+drive+system+in+the+human+disease+vector.">Development of a confinable gene drive system in the human disease vector.</a> eLife. 9, e51701 (2020).</li><li>Dalla Benetta, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+Antiviral+Sensor+Targets+Infected+Mosquitoes.">Engineered Antiviral Sensor Targets Infected Mosquitoes.</a> The CRISPR journal. 6 (6), 543-556 (2023).</li><li>Li, H. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-Type+lectins+link+immunological+and+reproductive+processes+in+Aedes+aegypti.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+3'+untranslated+region+of+baculovirus+p10+mRNA+in+high-level+expression+of+foreign+genes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+SV40+polyA+on+gene+expression+of+baculovirus+expression+vector+systems.">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

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&#39; and 3&#39; 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-EC...

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

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

625 Views.  University of California, San Diego. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

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

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

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

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

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

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

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R10 flow cell and Illumina sequencing as an example.

We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell.

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for ...

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

Summary

Explore More Videos

Chapters in this video

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

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

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example