Name: protocols for crispr/cas9 mutagenesis of the oriental fruit fly bactrocera dorsalis
Uploaded: 2022-09-28T12:40:00
Description: Watch this Scientific Journal Video about Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis at JoVE.com

Questo lavoro è stato sostenuto dal Programma di scienza e tecnologia di Shenzhen (sovvenzione n. KQTD20180411143628272) e fondi speciali per l'innovazione della tecnologia scientifica e lo sviluppo industriale del nuovo distretto di Shenzhen Dapeng (sovvenzione n. PT202101-02).

1. Progettazione del target e sintesi in vitro di sgRNA
<ol><li>Prevedere la struttura dei geni bersaglio di interesse e determinare i confini tra esoni e introni tramite l'analisi bioinformatica del genoma di B. dorsalis (le applicazioni software utilizzate qui sono elencate nella Tabella dei materiali). NOTA: BLAT20 è stato utilizzato per cercare potenziali loci genici nel genoma. Le letture di alta qualità dell'RNA-seq (trascr...

<ol>
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A., Charpentier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+frontier+of+genome+engineering+with+CRISPR-Cas9.">The new frontier of genome engineering with CRISPR-Cas9.</a> Science. 346 (6213), 1258096 (2014).</li><li>Cong, L., 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Li, 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=Heritable+gene+targeting+in+the+mouse+and+rat+using+a+CRISPR-Cas+system.">Heritable gene targeting in the mouse and rat using a CRISPR-Cas system.</a> Nature Biotechnology. 31 (8), 681-683 (2013).</li><li>Wang, 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=One-step+generation+of+mice+carrying+mutations+in+multiple+genes+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.</a> Cell. 153 (4), 910-918 (2013).</li><li>Li, 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=Generating+mutant+Aedes+aegypti+mosquitoes+using+the+CRISPR/Cas9+system.">Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system.</a> STAR protocols. 2 (2), 100432 (2021).</li><li>Zhu, G. -. 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=Expanding+the+toolkit+for+genome+editing+in+a+disease+vector,+Aedes+aegypti:+transgenic+lines+expressing+Cas9+and+single+guide+RNA+induce+efficient+mutagenesis.">Expanding the toolkit for genome editing in a disease vector, Aedes aegypti: transgenic lines expressing Cas9 and single guide RNA induce efficient mutagenesis.</a> The CRISPR Journal. 4 (6), 846-853 (2021).</li><li>Li, Y., 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=CRISPR/Cas9+in+locusts:+Successful+establishment+of+an+olfactory+deficiency+line+by+targeting+the+mutagenesis+of+an+odorant+receptor+co-receptor+(Orco).">CRISPR/Cas9 in locusts: Successful establishment of an olfactory deficiency line by targeting the mutagenesis of an odorant receptor co-receptor (Orco).</a> Insect Biochemistry and Molecular Biology. 79, 27-35 (2016).</li><li>Liu, Q., 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=Deletion+of+the+Bombyx+mori+odorant+receptor+co-receptor+(BmOrco)+impairs+olfactory+sensitivity+in+silkworms.">Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms.</a> Insect Biochemistry &amp; Molecular Biology. 86, 58-67 (2017).</li><li>Zhao, S., 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+somatic+and+germline+genome+engineering+of+Bactrocera+dorsalis+by+the+CRISPR/Cas9+system.">Efficient somatic and germline genome engineering of Bactrocera dorsalis by the CRISPR/Cas9 system.</a> Pest Management Science. 75 (7), 1921-1932 (2019).</li><li>Bai, X., 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=CRISPR/Cas9-mediated+knockout+of+the+eye+pigmentation+gene+white+leads+to+alterations+in+colour+of+head+spots+in+the+oriental+fruit+fly,+Bactrocera+dorsalis.">CRISPR/Cas9-mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis.</a> Insect Molecular Biology. 28 (6), 837-849 (2019).</li><li>Zheng, W., 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=Clustered+regularly+interspaced+short+palindromic+repeats+(CRISPR)/CRISPR-associated+9-mediated+mutagenesis+of+the+multiple+edematous+wings+gene+induces+muscle+weakness+and+flightlessness+in+Bactrocera+dorsalis+(Diptera:+Tephritidae).">Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9-mediated mutagenesis of the multiple edematous wings gene induces muscle weakness and flightlessness in Bactrocera dorsalis (Diptera: Tephritidae).</a> Insect Molecular Biology. 28 (2), 222-234 (2019).</li><li>Kent, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLAT-the+BLAST-like+alignment+tool.">BLAT-the BLAST-like alignment tool.</a> Genome Research. 12 (4), 656-664 (2002).</li><li>Kim, D., Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HISAT:+A+fast+spliced+aligner+with+low+memory+requirements.">HISAT: A fast spliced aligner with low memory requirements.</a> Nature Methods. 12 (4), 357-360 (2015).</li><li>Danecek, 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=Twelve+years+of+SAMtools+and+BCFtools.">Twelve years of SAMtools and BCFtools.</a> Gigascience. 10 (2), (2021).</li><li>Kovaka, S., 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=Transcriptome+assembly+from+long-read+RNA-seq+alignments+with+StringTie2.">Transcriptome assembly from long-read RNA-seq alignments with StringTie2.</a> Genome Biology. 20 (1), 278 (2019).</li><li>. TransDecoder Available from: <a target="_blank" href="https://github.com/TransDecoder/TransDecoder/releases/tag/TransDecoder-v5.5.0">https://github.com/TransDecoder/TransDecoder/releases/tag/TransDecoder-v5.5.0</a> (2018)</li><li>Robinson, J. 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=Integrative+genomics+viewer.">Integrative genomics viewer.</a> Nature Biotechnology. 29 (1), 24-26 (2011).</li><li>Chang, 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=A+pheromone+antagonist+regulates+optimal+mating+time+in+the+moth+Helicoverpa+armigera.">A pheromone antagonist regulates optimal mating time in the moth Helicoverpa armigera.</a> Current Biology. 27 (11), 1610-1615 (2017).</li><li>Xie, S., 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=sgRNAcas9:+a+software+package+for+designing+CRISPR+sgRNA+and+evaluating+potential+off-target+cleavage+sites.">sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites.</a> PloS One. 9 (6), 100448 (2014).</li><li>Zheng, Q. 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=Precise+gene+deletion+and+replacement+using+the+CRISPR/Cas9+system+in+human+cells.">Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells.</a> Biotechniques. 57 (3), 115-124 (2014).</li><li>Ren, L., 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=Rectal+bacteria+produce+sex+pheromones+in+the+male+oriental+fruit+fly.">Rectal bacteria produce sex pheromones in the male oriental fruit fly.</a> Current Biology. 31 (10), 2220-2226 (2021).</li><li>Xiao, Q., 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=Application+of+CRISPR/Cas9-based+gene+editing+in+HIV-1/AIDS+therapy.">Application of CRISPR/Cas9-based gene editing in HIV-1/AIDS therapy.</a> Frontiers in Cellular and Infection Microbiology. 9, 69 (2019).</li><li>El-Mounadi, 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=Principles,+applications,+and+biosafety+of+plant+genome+editing+using+CRISPR-Cas9.">Principles, applications, and biosafety of plant genome editing using CRISPR-Cas9.</a> Frontiers in Plant Science. 11, 56 (2020).</li><li>Hsu, P. 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=Development+and+applications+of+CRISPR-Cas9+for+genome+engineering.">Development and applications of CRISPR-Cas9 for genome engineering.</a> Cell. 157 (6), 1262-1278 (2014).</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 Research. 47, 171-174 (2019).</li><li>Ai, 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=Embryo+microinjection+and+knockout+mutant+identification+of+CRISPR/Cas9+genome-edited+Helicoverpa+armigera+(H&#252;bner).">Embryo microinjection and knockout mutant identification of CRISPR/Cas9 genome-edited Helicoverpa armigera (H&#252;bner).</a> Journal of Visualized Experiments. (173), e62068 (2021).</li><li>Xie, S., 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=sgRNAcas9:+a+software+package+for+designing+CRISPR+sgRNA+and+evaluating+potential+off-target+cleavage+sites.">sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites.</a> PloS One. 9 (6), 100448 (2014).</li><li>Gratz, S. 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=Genome+engineering+of+Drosophila+with+the+CRISPR+RNA-guided+Cas9+nuclease.">Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease.</a> Genetics. 194 (4), 1029-1035 (2013).</li><li>Zhu, G. 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=Knockout+of+juvenile+hormone+receptor,+Methoprene-tolerant,+induces+black+larval+phenotype+in+the+yellow+fever+mosquito,+Aedes+aegypti.">Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, Aedes aegypti.</a> Proceedings of the National Academy of Sciences. 116 (43), 21501-21507 (2019).</li><li>Laohakieat, K., Isasawin, S., Thanaphum, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transformer-2+and+fruitless+characterisation+with+developmental+expression+profiles+of+sex-determining+genes+in+Bactrocera+dorsalis+and+B.+correcta.">The transformer-2 and fruitless characterisation with developmental expression profiles of sex-determining genes in Bactrocera dorsalis and B. correcta.</a> Scientific Reports. 10 (1), 1-13 (2020).</li><li>Gabrieli, 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=Sperm-less+males+modulate+female+behaviour+in+Ceratitis+capitata+(Diptera:+Tephritidae).">Sperm-less males modulate female behaviour in Ceratitis capitata (Diptera: Tephritidae).</a> Insect Biochemistry and Molecular Biology. 79, 13-26 (2016).</li></ol>

Il sistema CRISPR / Cas9 è lo strumento di editing genetico più utilizzato e ha varie applicazioni, come il gene threpy30, il crop breeding 31 e gli studi di base sulle funzioni geniche32. Questo sistema è già stato applicato per l'editing genetico in varie specie di insetti ed è servito come strumento efficace per gli studi genetici funzionali nei parassiti. I protocolli che presentiamo qui standardizzano la procedura di progettazione e sintesi...

La mosca orientale della frutta, Bactrocera dorsalis , è una specie cosmopolita di insetti nocivi che causa danni a oltre 150 specie di colture da frutto, tra cui guava, mango, Eugenia spp., ciliegia del Suriname, agrumi, nespolo e papaia1. I danni causati nella sola provincia del Guangdong (Cina) sono stimati in oltre 200 milioni di yuan. Le femmine adulte inseriscono le loro uova sotto la buccia dei frutti maturi o maturi, causando il decadimento e l'abscissione del frutto, che diminuisce la qualità dei frutti e la resa complessiva del raccolto2. Poiché i moscerini della frutta adulti hanno una grande capacità di volo e le loro larve penetrano nella pelle del frutto, gli insetticidi che richiedono il contatto diretto con il parassita si comportano male sul campo. Inoltre, l'uso estensivo di insetticidi ha aumentato la resistenza di B. dorsalis contro vari prodotti chimici agricoli, rendendo ancora più difficile il controllo di questi parassiti dannosi3. Pertanto, lo sviluppo di strategie di gestione dei parassiti efficaci e rispettose dell'ambiente è disperatamente necessario.
Recentemente, con lo sviluppo di strumenti biologici molecolari e tecnologie di sequenziamento ad alto rendimento, gli scienziati stanno tentando di sviluppare strategie di gestione dei parassiti rispettose dell'ambiente, come RNAi, che mirano alla funzionalità di importanti geni (bersagli molecolari) di vari insetti nocivi. I geni fondamentali per la sopravvivenza e la riproduzione dell'organismo nocivo possono essere identificati mediante studi genetici funzionali e fungere ulteriormente da potenziali bersagli molecolari per il miglioramento di strumenti di gestione fitosanitaria specificamente mirati e rispettosi dell'ambiente4. Per adattare tali strategie al controllo del moscerino della frutta orientale, sono necessari metodi efficaci per la ricerca genica funzionale.
Il sistema endonucleasico CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) è stato inizialmente scoperto nei batteri e negli archei e si è scoperto essere un meccanismo adattativo coinvolto nel riconoscimento e nella degradazione del DNA intracellulare estraneo, come quello introdotto dall'infezione dei batteriofagi5. Nel sistema CRISPR di tipo II, l'endonucleasi Cas9 è guidata da piccoli RNA associati (crRNA e tracrRNA) per scindereil DNA 6,7,8 ed è diventato uno degli strumenti più utilizzati per l'editing genetico fino ad oggi9,10,11,12. Poiché il sistema CRISPR / Cas9 ha diversi vantaggi, come l'alta efficienza del silenziamento genico e il basso costo, è già stato applicato per l'editing genetico in varie specie di insetti, tra cui Aedes aegypti13,14, Locusta migratoria15 e Bombyx mori16. In B. dorsalis, i geni relativi al colore del corpo, al dimorfismo delle ali e alla determinazione del sesso sono stati eliminati con successo usando CRISPR / Cas917,18,19. Tuttavia, le procedure dettagliate per l'applicazione di CRISPR / Cas9 in questo insetto rimangono incomplete. Inoltre, alcuni passaggi forniti dai ricercatori per l'editing genetico di B. dorsalis sono anche vari e necessitano di standardizzazione. Ad esempio, le forme di Cas9 erano diverse nei riferimenti pubblicati17,18,19.
Questo articolo fornisce un metodo sistematico per la mutagenesi di B. dorsalis utilizzando il sistema CRISPR / Cas9, compresa la progettazione e la sintesi di RNA guida, la raccolta di embrioni, l'iniezione di embrioni, l'allevamento di insetti e lo screening mutante. Questo protocollo servirà come guida utile per generare mosche mutanti per i ricercatori che sono interessati agli studi sui geni funzionali in B. dorsalis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6x DNA Loading Buffer</td>
			<td>TransGen Biotech</td>
			<td>GH101-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial climate chamber</td>
			<td>ShangHai BluePard</td>
			<td>MGC-350P</td>
			<td></td>
		</tr>
		<tr>
			<td>AxyPrep Genomic DNA Mini-Extraction Kit</td>
			<td>Axygen</td>
			<td>AP-MN-MS-GDNA-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>BLAT</td>
			<td>NA</td>
			<td>NA</td>
			<td>For searching potential gene loci in the genome</td>
		</tr>
		<tr>
			<td>Capillary Glass</td>
			<td>WPI&#160;</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf InjectMan 4 micromanipulator</td>
			<td>Eppendorf</td>
			<td>InjectMan 4</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneArt Precision gRNA Synthesis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A29377</td>
			<td></td>
		</tr>
		<tr>
			<td>Hisat2</td>
			<td>NA</td>
			<td>NA</td>
			<td>For aligning the transcriptome to the acquired gene loci</td>
		</tr>
		<tr>
			<td>IGV</td>
			<td>NA</td>
			<td>NA</td>
			<td>For visualizing the results from Transdecoder</td>
		</tr>
		<tr>
			<td>Microgrinder</td>
			<td>NARISHIGE</td>
			<td>EG-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZ2-ILST</td>
			<td></td>
		</tr>
		<tr>
			<td>pEASY-Blunt Cloning Kit</td>
			<td>TransGen Biotech</td>
			<td>CB101-02</td>
			<td>https://www.transgenbiotech.com/data/upload/pdf/CB101_2022-07-14.pdf</td>
		</tr>
		<tr>
			<td>Phenol red solution</td>
			<td>Sigma-Aldrich</td>
			<td>P0290-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette cookbook 2018 P-97 &#38; P-1000 Micropipette Pullers</td>
			<td>Instrument Company&#160;</td>
			<td></td>
			<td>https://www.sutter.com/PDFs/cookbook.pdf</td>
		</tr>
		<tr>
			<td>PrimeSTAR HS (Premix)</td>
			<td>Takara Biomedical Technology</td>
			<td>R040A</td>
			<td></td>
		</tr>
		<tr>
			<td>SAMtools</td>
			<td>NA</td>
			<td>NA</td>
			<td>For generating the sorted bam files</td>
		</tr>
		<tr>
			<td>sgRNAcas9-AI</td>
			<td>NA</td>
			<td>NA</td>
			<td>sgRNA design 
			http://123.57.239.141:8080/home</td>
		</tr>
		<tr>
			<td>Sutter Micropipette Puller Sutter&#160;</td>
			<td>Instrument Company&#160;</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans2K DNA Marker</td>
			<td>TransGen Biotech</td>
			<td>BM101-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Transdecoder</td>
			<td>NA</td>
			<td>NA</td>
			<td>For combining the results of assemble transcripts and gene loci information 
			https://github.com/TransDecoder/TransDecoder/releases/tag/TransDecoder-v5.5.0</td>
		</tr>
		<tr>
			<td>TrueCut Cas9 Protein v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>A36498</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-trace biological detector</td>
			<td>Thermo Fisher Scientific</td>
			<td>Nanodrop 2000C</td>
			<td></td>
		</tr>
	</tbody>
</table>

protocols for crispr/cas9 mutagenesis of the oriental fruit fly bactrocera dorsalis

La mosca della frutta orientale, Bactrocera dorsalis, è una specie di parassiti altamente invasiva e adattativa che causa danni agli agrumi e ad oltre 150 altre colture da frutto in tutto il mondo. Poiché i moscerini della frutta adulti hanno una grande capacità di volo e le femmine depongono le uova sotto le bucce della frutta, gli insetticidi che richiedono il contatto diretto con il parassita di solito funzionano male sul campo. Con lo sviluppo di strumenti biologici molecolari e la tecnologia di sequenziamento ad alto rendimento, molti scienziati stanno tentando di sviluppare strategie di gestione dei parassiti rispettose dell'ambiente. Questi includono RNAi o pesticidi basati sull'editing genetico che sottoregolano o silenziano i geni (bersagli molecolari), come i geni olfattivi coinvolti nel comportamento di ricerca, in vari insetti nocivi. Per adattare queste strategie per il controllo del moscerino della frutta orientale, sono necessari metodi efficaci per la ricerca genica funzionale. I geni con funzioni critiche nella sopravvivenza e nella riproduzione di B. dorsalis servono come buoni bersagli molecolari per l'abbattimento e/o il silenziamento dei geni. Il sistema CRISPR / Cas9 è una tecnica affidabile utilizzata per l'editing genetico, specialmente negli insetti. Questo articolo presenta un metodo sistematico per la mutagenesi CRISPR/Cas9 di B. dorsalis, compresa la progettazione e la sintesi di RNA guida, la raccolta di embrioni, l'iniezione di embrioni, l'allevamento di insetti e lo screening mutante. Questi protocolli serviranno come guida utile per generare mosche mutanti per i ricercatori interessati agli studi sui geni funzionali in B. dorsalis.

Questo protocollo presenta passaggi dettagliati per lo sviluppo di mutanti di B. dorsalis utilizzando la tecnologia CRISPR / Cas9, compresi i risultati rappresentativi della selezione del gDNA, la raccolta di embrioni e microiniezione, il mantenimento degli insetti e lo screening dei mutanti.
L'esempio del sito bersaglio del gene selezionato si trova nel terzo esone (Figura 1C). Questo sito è altamente conservato e una singola banda è stata rilevata med...

Watch this Scientific Journal Video about Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis at JoVE.com

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

Questo articolo presenta i protocolli passo-passo per la mutagenesi CRISPR/Cas9 del moscerino orientale della frutta Bactrocera dorsalis. I passaggi dettagliati forniti da questo protocollo standardizzato serviranno come guida utile per generare mosche mutanti per studi genetici funzionali in B. dorsalis.

Protocolli per la mutagenesi CRISPR/Cas9 del moscerino orientale della frutta Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...

Il moscerino orientale della frutta è una specie di parassiti altamente invasiva e adattabile. Metodi efficaci per la ricerca genetica funzionale potrebbero aiutarci a sviluppare strategie di gestione dei parassiti rispettose dell'ambiente. Questo metodo è ad alta efficienza e basso costo.Il nostro protocollo servirà come guida utile per generare mosche mutanti per i ricercatori di moscerini della frutta orientali. Per iniziare, prevedere la struttura dei geni bersaglio di interesse e determinare i confini tra esoni e introni attraverso l'analisi bioinformatica del genoma di Bactroceras dorsalis. Utilizzare il kit di sintesi del gRNA disponibile in commercio per generare lo sgRNA progettato.Risospendere il prodotto di gRNA in acqua priva di nucleasi. Quantificare la concentrazione utilizzando uno spettrofotometro UV-visibile e conservare a 80 gradi Celsius prima dell'uso. Metti le pupe di B.dorsalis in una gabbia di plastica, con una condizione di allevamento del 55% di umidità relativa, 26,5 gradi Celsius e un ciclo di luce / giorno 14:10.Quando gli adulti si chiudono, fornire una miscela di zucchero e lievito come cibo insieme all'acqua. La maggior parte degli adulti raggiunge la maturità sessuale 10 giorni dopo l'emergenza. Utilizzare un supporto luminoso con una luce da 30 a 50 lux per migliorare la fecondità delle femmine, migliorando così l'efficienza della raccolta degli embrioni.Quindi, posizionare una garza a 200 maglie nella camera di ovideposizione, a circa 1 o 2 millimetri di distanza dal coperchio della camera. Inserire una nuova camera di ovodeposizione nella gabbia quando la configurazione della microiniezione è pronta. Raccogliere gli embrioni ogni 10 minuti usando una spazzola bagnata fine e allinearli su una piastra di iniezione fatta da sé.Immergere gli embrioni in olio di alocarbonio durante l'iniezione per evitare un ulteriore disseccamento. Preparare la soluzione di lavoro mescolando la proteina Cas9 e il corrispondente sgRNA ad una concentrazione di 300 nanogrammi per microlitri di ciascuno. Quindi, aggiungere 1 microlitro di rosso fenolo alla miscela per servire come un modo conveniente per contrassegnare gli embrioni iniettati.Posizionare la miscela preparata su ghiaccio per evitare la degradazione dello sgRNA. Preparare l'ago per iniezione di vetro utilizzando un estrattore di micropipette. Aggiungere 3 microlitri della miscela nell'ago per iniezione senza introdurre bolle d'aria.Quindi, aprire l'ago utilizzando un Microgrinder e collegarlo al micromanipolatore secondo la guida per l'utente. Impostare i parametri per l'iniettore come Pi su 500 ettopascal. Ti a 0,5 secondi e PC a 200 ettopascal.Posizionare il piatto con gli embrioni allineati sul tavolo dell'obiettivo. Quindi, regolare la posizione della micropipetta sotto un microscopio ottico fine, impostando la micropipetta e l'embrione sullo stesso piano. Inserire la punta dell'ago nella parte posteriore, o polo vegetale, dell'embrione.Quindi, premere il pedale dall'iniettore per consegnare le miscele nell'embrione. L'aspetto di un leggero colore rossastro è dovuto all'aggiunta di rosso fenolo nella miscela. Posizionare la piastra di iniezione con gli embrioni iniettati in una camera climatica artificiale.Dopo 36 ore, trasferire le larve tratteggiate nella dieta larvale usando un pennello a punta fine. Raccogli le larve tre o quattro volte al giorno fino a quando non si schiudono più larve. Quindi, posizionare le larve mature nella sabbia bagnata per impuparsi.La fase di pupa dura 10 giorni. Dopo la pupa, spostare le pupe in una gabbia di plastica prima che inizi l'eclosione. Dopo aver isolato il DNA genomico da un pupario fresco o da una singola gamba centrale di singoli adulti e aver progettato i primer specifici per amplificare l'area target, eseguire la reazione a catena della polimerasi, o PCR, seguendo le condizioni cicliche descritte nel manoscritto testuale.Quindi, sequenziare i prodotti PCR purificati. Il rilevamento di più picchi sovrapposti adiacenti al sito bersaglio suggerisce una mutagenesi riuscita. Per la sub-clonazione, mescolare i prodotti PCR purificati con un vettore smussato e ligarli a 25 gradi Celsius per 15 minuti.Quindi, aggiungere le cellule trans-T1 e metterlo sul ghiaccio per 30 minuti. Quindi, aggiungere 500 microlitri di mezzo LB e incubare a 37 gradi Celsius per 1 ora agitando. Centrifugare e scartare il surnatante.Risospendere il pellet e stenderlo su una piastra LB. Posizionare la piastra per l'incubazione notturna a 37 gradi Celsius. Quindi, selezionare le singole colonie batteriche per il sequenziamento per verificare il genotipo dei mutanti.In questo studio, l'esempio del sito bersaglio del gene selezionato si trova nel terzo esone. Il sito bersaglio è altamente conservato e una singola banda è stata rilevata mediante elettroforesi su gel per il modello di DNA per gRNA sintetico e gRNA ottenuto in trascrizione in vitro. La sopravvivenza e la mutagenesi di B.dorsalis dopo microiniezioni sono mostrate qui.Dopo il sequenziamento dei prodotti PCR, l'80% degli individui G0 sono mutanti a mosaico. Nello screening mutante, il mutante con delezione di 8 coppie di basi ha provocato la cessazione prematura della traduzione di aminoacidi. L'inserimento dell'ago nella zona posteriore o nel polo vegetale di un embrione è un passo centrale perché influisce direttamente sulla sopravvivenza delle uova.Questa tecnologia fornisce un riferimento per lo studio delle aree delle funzioni geniche in B.Dorsalis Possono catturare rapidamente i mutanti che vogliono attraverso questo metodo.

protocols-for-crisprcas9-mutagenesis-oriental-fruit-fly-bactrocera

Research

JoVE Journal

Biology

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

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Protocolli di selezione mutagenesi e funzionale per l&#39;evoluzione Regia di proteine ​​in E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Ricerca

Biologia

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

Isolamento e studio della biofisica cuticole di frutta

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

Generazione di Genomic eliminazioni in linee cellulari di mammiferi via CRISPR / Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Silenziamento della scintilla: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

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

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

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

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.

Preparazione e iniezione di embrioni di zanzare Culex per generare mutazioni nulle utilizzando CRISPR / Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

Ingegneria genomica delle cellule B umane primarie utilizzando CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

Costruzione di mutanti omozigoti di locuste migratorie utilizzando la tecnologia CRISPR/Cas9

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

CRISPR/Cas9 Gene Editing di cellule staminali e progenitrici ematopoietiche per applicazioni di terapia genica

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

Modifiche precise mediate da CRISPR-Cas9 nei cuori di zebrafish

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...

Editing genetico della proteina-E associata al centromero utilizzando il sistema CRISPR/Cas9

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system.
Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.
In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E&#160;knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

Autore in primo piano: Stabilire le cellule HeLa knockout CENP-E - Un nuovo approccio per studiare la biologia CENP-E della chinesina-7 e dei suoi inibitori

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E&#160;knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

Generazione di linee cellulari knockout CENP-E-/- associate al centromero della proteina E utilizzando il sistema CRISPR/Cas9

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...