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

Este trabajo fue apoyado por el Programa de Ciencia y Tecnología de Shenzhen (Subvención No. KQTD20180411143628272) y fondos especiales para la innovación de tecnología científica y el desarrollo industrial del Nuevo Distrito de Shenzhen Dapeng (Subvención No. PT202101-02).

1. Diseño de diana y síntesis in vitro de sgRNA
<ol><li>Predecir la estructura de los genes diana de interés y determinar los límites entre exones e intrones mediante el análisis bioinformático del genoma de B. dorsalis (las aplicaciones de software utilizadas aquí se enumeran en la Tabla de materiales). NOTA: BLAT20 se utilizó para buscar posibles loci de genes en el genoma. Las lecturas de RNA-seq de alta calidad (transcr...

<ol>
	<li>Christenson, L. D., Foote, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+fruit+flies.">Biology of fruit flies.</a> Annual Review of Entomology. 5 (1), 171-192 (1960).</li><li>Ma, 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=The+assessment+of+the+economic+losses+caused+by+Bactrocera+dorsalis,+B.+cucurbitae+and+B.+tau+to+Guangdong+province.">The assessment of the economic losses caused by Bactrocera dorsalis, B. cucurbitae and B. tau to Guangdong province.</a> Plant Quarantine. 27 (3), 50-56 (2013).</li><li>Jin, 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=Chemical+control+measures+and+drug+resistance+management+of+Bactrocera+dorsalis.">Chemical control measures and drug resistance management of Bactrocera dorsalis.</a> Agrochemicals. 60 (1), 1-5 (2021).</li><li>Yang, B., Liu, Y., Wang, B., Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction-based+behaviorally+manipulated+technology+of+pest+insects:+research+progress,+opportunities+and+challenges.">Olfaction-based behaviorally manipulated technology of pest insects: research progress, opportunities and challenges.</a> Bulletin of National Natural Science Foundation of China. 34 (4), 441-446 (2020).</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>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>Pyzocha, N. 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=RNA-guided+genome+editing+of+mammalian+cells.">RNA-guided genome editing of mammalian cells.</a> Gene Correction. , 269-277 (2014).</li><li>Weiss, 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=CRISPR-Cas+systems:+new+players+in+gene+regulation+and+bacterial+physiology.">CRISPR-Cas systems: new players in gene regulation and bacterial physiology.</a> Frontiers in Cellular and Infection Microbiology. 4, 37 (2014).</li><li>Doudna, J. 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>

El sistema CRISPR/Cas9 es la herramienta de edición de genes más utilizada y tiene varias aplicaciones, como la trepida 30, el mejoramiento de cultivos31 y los estudios básicos de funciones genéticas32. Este sistema ya se ha aplicado para la edición de genes en varias especies de insectos y ha servido como una herramienta eficaz para el estudio de genes funcionales en plagas. Los protocolos que presentamos aquí estandarizan el procedimiento de ...

La mosca oriental de la fruta, Bactrocera dorsalis , es una especie cosmopolita de plaga de insectos que causa daños a más de 150 especies de cultivos frutales, incluyendo guayaba, mango, Eugenia spp., cereza de Surinam, cítricos, níspero y papaya1. Los daños causados solo en la provincia de Guangdong (China) se estiman en más de 200 millones de yuanes. Las hembras adultas insertan sus huevos debajo de la piel de los frutos maduros o maduros, causando caries y abscisión del fruto, lo que disminuye la calidad del fruto y el rendimiento general del cultivo2. Dado que las moscas adultas de la fruta tienen una gran capacidad de vuelo y sus larvas perforan la piel de la fruta, los insecticidas que requieren contacto directo con la plaga funcionan mal en el campo. Además, el uso extensivo de insecticidas ha aumentado la resistencia de B. dorsalis contra diversos productos químicos agrícolas, lo que dificulta aún más el control de estas plagas dañinas3. Por lo tanto, se necesita desesperadamente el desarrollo de estrategias de manejo de plagas efectivas y respetuosas con el medio ambiente.
Recientemente, con el desarrollo de herramientas biológicas moleculares y tecnologías de secuenciación de alto rendimiento, los científicos están tratando de desarrollar estrategias de manejo de plagas respetuosas con el medio ambiente, como el ARNi, que se dirigen a la funcionalidad de genes importantes (objetivos moleculares) de varias plagas de insectos. Los genes que son críticos para la supervivencia y reproducción de la plaga pueden identificarse a través de estudios genéticos funcionales y además sirven como posibles objetivos moleculares para la mejora de herramientas de manejo de plagas específicamente específicas y respetuosas con el medio ambiente4. Para adaptar tales estrategias al control de la mosca oriental de la fruta, se necesitan métodos efectivos para la investigación genética funcional.
El sistema de endonucleasa CRISPR/Cas (clustered regular interspaced short palindromic repeats/CRISPR-associated) fue descubierto inicialmente en bacterias y arqueas y resultó ser un mecanismo adaptativo implicado en el reconocimiento y degradación del ADN intracelular extraño, como el introducido por bacteriófagos infectantes5. En el sistema CRISPR tipo II, la endonucleasa Cas9 es guiada por pequeños ARN asociados (crRNA y tracrRNA) para escindirel ADN 6,7,8 y se ha convertido en una de las herramientas más utilizadas para la edición de genes hasta la fecha9,10,11,12. Dado que el sistema CRISPR / Cas9 tiene varias ventajas, como la alta eficiencia del silenciamiento génico y el bajo costo, ya se ha aplicado para la edición de genes en varias especies de insectos, incluyendo Aedes aegypti13,14, Locusta migratoria15 y Bombyx mori16. En B. dorsalis, los genes relacionados con el color del cuerpo, el dimorfismo del ala y la determinación del sexo han sido eliminados con éxito utilizando CRISPR / Cas917,18,19. Sin embargo, los procedimientos detallados para la aplicación de CRISPR/Cas9 en este insecto siguen siendo incompletos. Además, algunos pasos proporcionados por los investigadores para la edición de genes de B. dorsalis también son variados y necesitan estandarización. Por ejemplo, las formas de Cas9 fueron diferentes en las referencias publicadas17,18,19.
Este documento proporciona un método sistemático para la mutagénesis de B. dorsalis utilizando el sistema CRISPR / Cas9, incluido el diseño y la síntesis de ARN guía, la recolección de embriones, la inyección de embriones, la cría de insectos y la detección de mutantes. Este protocolo servirá como una guía útil para generar moscas mutantes para los investigadores que estén interesados en los estudios de genes funcionales en 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 oriental de la fruta, Bactrocera dorsalis, es una especie de plaga altamente invasiva y adaptativa que causa daños a los cítricos y a más de 150 cultivos frutales en todo el mundo. Dado que las moscas adultas de la fruta tienen una gran capacidad de vuelo y las hembras ponen sus huevos debajo de las pieles de la fruta, los insecticidas que requieren contacto directo con la plaga generalmente funcionan mal en el campo. Con el desarrollo de herramientas de biología molecular y tecnología de secuenciación de alto rendimiento, muchos científicos están tratando de desarrollar estrategias de manejo de plagas respetuosas con el medio ambiente. Estos incluyen ARNi o pesticidas basados en la edición de genes que regulan a la baja o silencian los genes (objetivos moleculares), como los genes olfativos involucrados en el comportamiento de búsqueda, en varias plagas de insectos. Para adaptar estas estrategias para el control de la mosca oriental de la fruta, se necesitan métodos efectivos para la investigación genética funcional. Los genes con funciones críticas en la supervivencia y reproducción de B. dorsalis sirven como buenos objetivos moleculares para la eliminación y / o silenciamiento de genes. El sistema CRISPR/Cas9 es una técnica fiable utilizada para la edición de genes, especialmente en insectos. Este artículo presenta un método sistemático para la mutagénesis CRISPR / Cas9 de B. dorsalis, incluido el diseño y la síntesis de ARN guía, la recolección de embriones, la inyección de embriones, la cría de insectos y la detección de mutantes. Estos protocolos servirán como una guía útil para generar moscas mutantes para los investigadores interesados en estudios de genes funcionales en B. dorsalis.

Este protocolo presenta pasos detallados para el desarrollo de mutantes de B. dorsalis utilizando la tecnología CRISPR / Cas9, incluidos los resultados representativos de la selección de ADNg, la recolección de embriones y microinyección, el mantenimiento de insectos y la detección de mutantes.
El ejemplo del sitio objetivo del gen seleccionado se encuentra en el tercer exón (Figura 1C). Este sitio está altamente conservado, y se detectó una sola ...

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

Este artículo presenta los protocolos paso a paso para la mutagénesis CRISPR/Cas9 de la mosca oriental de la fruta Bactrocera dorsalis. Los pasos detallados proporcionados por este protocolo estandarizado servirán como una guía útil para generar moscas mutantes para estudios de genes funcionales en B. dorsalis.

Protocolos para la mutagénesis CRISPR/Cas9 de la mosca oriental de la fruta 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 ...

La mosca oriental de la fruta es una especie plaga altamente invasiva y adaptativa. Los métodos efectivos para la investigación genética funcional podrían ayudarnos a desarrollar estrategias de manejo de plagas respetuosas con el medio ambiente. Este método es de alta eficiencia y bajo costo.Nuestro protocolo servirá como una guía útil para generar moscas mutantes para los investigadores de moscas orientales de la fruta. Para empezar, predecir la estructura de los genes diana de interés y determinar los límites entre exones e intrones mediante el análisis bioinformático del genoma de Bactroceras dorsalis. Utilice el kit de síntesis de ARNg disponible comercialmente para generar el ARNg diseñado.Resuspender el producto de ARNg en agua libre de nucleasas. Cuantifique la concentración utilizando un espectrofotómetro UV-visible y guárdela a 80 grados centígrados antes de su uso. Coloque las pupas de B.dorsalis en una jaula de plástico, con una condición de crianza del 55% de humedad relativa, 26.5 grados centígrados y un ciclo de luz / día de 14:10.Cuando los adultos cierren, proporcione una mezcla de azúcar y levadura como alimento junto con agua. La mayoría de los adultos alcanzan la madurez sexual 10 días después de la emergencia. Utilice un soporte de luz con una luz de 30 a 50 lux para mejorar la fecundidad de las hembras, por lo tanto, mejorar la eficiencia de la recolección de embriones.Luego, coloque una gasa de malla 200 en la cámara de oviposición, aproximadamente de 1 a 2 milímetros de distancia de la tapa de la cámara. Coloque una nueva cámara de oviposición en la jaula cuando la configuración de microinyección esté lista. Recoja los embriones cada 10 minutos con un cepillo fino y húmedo y alinearlos en una placa de inyección hecha por él mismo.Sumerja los embriones en aceite de halocarbono durante la inyección para evitar una mayor desecación. Preparar la solución de trabajo mezclando la proteína Cas9 y el sgRNA correspondiente a una concentración de 300 nanogramos por microlitros de cada uno. Luego, agregue 1 microlitro de rojo fenol a la mezcla para que sirva como una forma conveniente de marcar los embriones inyectados.Coloque la mezcla preparada en hielo para evitar la degradación del sgRNA. Prepare la aguja de inyección de vidrio con un extractor de micropipetas. Añadir 3 microlitros de la mezcla en la aguja de inyección sin introducir burbujas de aire.Luego, abra la aguja con un Microgrinder y conéctela al micromanipulador de acuerdo con la guía del usuario. Establezca los parámetros para el inyector como Pi a 500 hectopascales. Ti a 0,5 segundos, y PC a 200 hectopascales.Coloque el plato con embriones alineados en la mesa objetivo. A continuación, ajuste la posición de la micropipeta bajo un microscopio óptico fino, colocando la micropipeta y el embrión en el mismo plano. Inserte la punta de la aguja en el polo posterior, o vegetal, del embrión.Luego, presione el pedal del inyector para administrar las mezclas en el embrión. La aparición de un ligero color rojizo se debe a la adición de rojo fenol en la mezcla. Coloque la placa de inyección con los embriones inyectados en una cámara climática artificial.Después de 36 horas, transfiera las larvas eclosionadas a la dieta larvaria con un cepillo de punta fina. Recoja las larvas tres o cuatro veces al día hasta que no eclosionen más larvas. A continuación, coloque las larvas maduras en la arena húmeda para pupar.La etapa de pupación dura 10 días. Después de la pupación, mueva las pupas a una jaula de plástico antes de que comience la eclosión. Después de aislar el ADN genómico de un pupario fresco o de una sola pierna media de adultos individuales y diseñar los cebadores específicos para amplificar el área objetivo, realice la reacción en cadena de la polimerasa, o PCR, siguiendo las condiciones de ciclo descritas en el texto manuscrito.Luego, secuencie los productos de PCR purificados. La detección de múltiples picos superpuestos adyacentes al sitio objetivo sugiere una mutagénesis exitosa. Para la subclonación, mezcle los productos de PCR purificados con un vector romo y ligarlos a 25 grados centígrados durante 15 minutos.Luego, agregue células trans-T1 y colóquelo en hielo durante 30 minutos. Luego, agregue 500 microlitros de LB medio e incube a 37 grados centígrados durante 1 hora con agitación. Centrifugar y desechar el sobrenadante.Vuelva a suspender el pellet y extiéndalo en una placa LB. Coloque la placa para la incubación durante la noche a 37 grados centígrados. Luego, seleccione colonias bacterianas individuales para la secuenciación para verificar el genotipo de los mutantes.En este estudio, el ejemplo del sitio objetivo del gen seleccionado se encuentra en el tercer exón. El sitio objetivo está altamente conservado, y se detectó una sola banda mediante electroforesis en gel para la plantilla de ADN para ARNg sintético y ARNg obtenido en transcripción in vitro. La supervivencia de B.dorsalis y la mutagénesis después de las microinyecciones se muestran aquí.Después de secuenciar los productos de PCR, el 80% de los individuos G0 son mutantes en mosaico. En la detección de mutantes, el mutante con deleción de 8 pares de bases resultó en la terminación prematura de la traducción de aminoácidos. Insertar la aguja en el área posterior o polo vegetal de un embrión es un paso central porque esto afecta directamente la supervivencia de los óvulos.Esta tecnología proporciona una referencia para estudiar áreas de funciones génicas en B.Dorsalis que pueden capturar rápidamente los mutantes que desean a través de este método.

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.

Mutagénesis y funcional protocolos de selección de la evolución dirigida de proteínas en 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 ...

Investigación

Biología

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.

El aislamiento y estudio biofísico de las cutículas de fruta

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.

Generación de deleciones genómicas en líneas celulares de mamíferos a través de 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.

Silenciar la chispa: Edición del genoma CRISPR/Cas9 en peces débilmente eléctricos

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.

Preparación e inyección de embriones de mosquitos Culex para generar mutaciones nulas utilizando 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.

Ingeniería genómica de células B humanas primarias usando 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.

Construcción de mutantes homocigotos de langostas migratorias utilizando tecnología 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.

Edición génica CRISPR/Cas9 de células madre y progenitoras hematopoyéticas para aplicaciones de terapia génica

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.

Ediciones precisas mediadas por CRISPR-Cas9 en corazones de pez cebra

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

Edición génica de la proteína-E asociada al centrómero mediante el 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.

Autor destacado: Establecimiento de células HeLa knockout de CENP-E : un enfoque novedoso para estudiar la biología de la kinesina-7 CENP-E y sus inhibidores

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.

Generación de líneas celulares knockout de proteína E CENP-E-/- asociadas al centrómero utilizando el 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 ...