Name: crispr gene editing tool for microrna cluster network analysis
Uploaded: 2022-04-25T13:00:00
Description: Watch this Scientific Journal Video about CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis at JoVE.com

पीसी 3-एमएल सेल लाइनों को मार्क स्टीयरन (ड्रेक्सल यूनिवर्सिटी कॉलेज ऑफ मेडिसिन) द्वारा प्रदान किया गया था। जस्टिन टोक्सी ने पीसीआर जीनोटाइपिंग में सहायता की। इस काम को ब्रीडन एडम्स फाउंडेशन ग्रांट, एक रयान ट्रांसलेशनल रिसर्च फंड और एई-के के लिए एक राष्ट्रमंडल स्वास्थ्य अनुसंधान बोर्ड अनुदान (सीएचआरबी -274-11-20) द्वारा समर्थित किया गया था।

1. सीआरआईएसपीआर जीन संपादन के लिए तैयारी और एमआईआरएनए क्लस्टर नॉकआउट सेल लाइनों को उत्पन्न करने के लिए आरएनए डिजाइन का मार्गदर्शन करें
<ol><li>एक डीएनए अनुक्रम एनोटेशन सॉफ्टवेयर प्रोग्राम19</...

<ol>
	<li>Hasegawa, T., Lewis, H., Esquela-Kerscher, A., Laurence, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Translating MicroRNAs to the Clinic. , 329-369 (2017).</li><li>Lewis, H., Esquela-Kerscher, A., Thiagalingam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Systems Biology of Cancer. , 134-153 (2015).</li><li>Esquela-Kerscher, A., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncomirs+-+microRNAs+with+a+role+in+cancer.">Oncomirs - microRNAs with a role in cancer.</a> Nature Reviews Cancer. 6 (4), 259-269 (2006).</li><li>Breving, K., Esquela-Kerscher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complexities+of+microRNA+regulation:+mirandering+around+the+rules.">The complexities of microRNA regulation: mirandering around the rules.</a> The International Journal of Biochemistry &amp; Cell Biology. 42 (8), 1316-1329 (2010).</li><li>Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+tools+for+microRNA+genomics.">miRBase: tools for microRNA genomics.</a> Nucleic Acids Research. 36, 154-158 (2008).</li><li>Kabekkodu, S. 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=Clustered+miRNAs+and+their+role+in+biological+functions+and+diseases.">Clustered miRNAs and their role in biological functions and diseases.</a> Biological Reviews. 93 (4), 1955-1986 (2018).</li><li>Xiang, J., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feud+or+friend?+The+role+of+the+miR-17-92+cluster+in+tumorigenesis.">Feud or friend? The role of the miR-17-92 cluster in tumorigenesis.</a> Current Genomics. 11 (2), 129-135 (2010).</li><li>Aqeilan, R. I., Calin, G. A., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-15a+and+miR-16-1+in+cancer:+discovery,+function+and+future+perspectives.">miR-15a and miR-16-1 in cancer: discovery, function and future perspectives.</a> Cell Death &amp; Differentiation. 17 (2), 215-220 (2010).</li><li>Hasegawa, 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=Characterization+and+evidence+of+the+miR-888+cluster+as+a+novel+cancer+network+in+prostate.">Characterization and evidence of the miR-888 cluster as a novel cancer network in prostate.</a> Molecular Cancer Research. , (2018).</li><li>Lewis, 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=miR-888+is+an+expressed+prostatic+secretions-derived+microRNA+that+promotes+prostate+cell+growth+and+migration.">miR-888 is an expressed prostatic secretions-derived microRNA that promotes prostate cell growth and migration.</a> Cell Cycle. 13 (2), 227-239 (2014).</li><li>Xu, 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=Evidence+for+a+prostate+cancer+susceptibility+locus+on+the+X+chromosome.">Evidence for a prostate cancer susceptibility locus on the X chromosome.</a> Nature Genetics. 20 (2), 175-179 (1998).</li><li>Schleutker, 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=A+genetic+epidemiological+study+of+hereditary+prostate+cancer+(HPC)+in+Finland:+frequent+HPCX+linkage+in+families+with+late-onset+disease.">A genetic epidemiological study of hereditary prostate cancer (HPC) in Finland: frequent HPCX linkage in families with late-onset disease.</a> Clinical Cancer Research. 6 (12), 4810-4815 (2000).</li><li>Farnham, J. M., Camp, N. J., Swensen, J., Tavtigian, S. V., Albright, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+the+HPCX+prostate+cancer+predisposition+locus+in+large+Utah+prostate+cancer+pedigrees.">Confirmation of the HPCX prostate cancer predisposition locus in large Utah prostate cancer pedigrees.</a> Human Genetics. 116 (3), 179-185 (2005).</li><li>Brown, W. 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=Hereditary+prostate+cancer+in+African+American+families:+linkage+analysis+using+markers+that+map+to+five+candidate+susceptibility+loci.">Hereditary prostate cancer in African American families: linkage analysis using markers that map to five candidate susceptibility loci.</a> British Journal Of Cancer. 90 (2), 510-514 (2004).</li><li>Bochum, 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=Confirmation+of+the+prostate+cancer+susceptibility+locus+HPCX+in+a+set+of+104+German+prostate+cancer+families.">Confirmation of the prostate cancer susceptibility locus HPCX in a set of 104 German prostate cancer families.</a> Prostate. 52 (1), 12-19 (2002).</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=Genome+editing.+The+new+frontier+of+genome+engineering+with+CRISPR-Cas9.">Genome editing. The new frontier of genome engineering with CRISPR-Cas9.</a> Science. 346 (6213), 1258096 (2014).</li><li>Adli, M. <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+tool+kit+for+genome+editing+and+beyond.">The CRISPR tool kit for genome editing and beyond.</a> Nature Communications. 9 (1), (2018).</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>. SnapGene&#174; software (from Insightful Science; available at snapgene.com) Available from: <a target="_blank" href="https://www.snapgene.com">https://www.snapgene.com</a> (2022)</li><li>. Ensembl Release 104 genome browser (from EMBL-EBI) Available from: <a target="_blank" href="https://www.ensembl.org">https://www.ensembl.org</a> (2022)</li><li>Howe, K. 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=Ensembl+2021.">Ensembl 2021.</a> Nucleic Acids Research. 49, 884-891 (2021).</li><li>. miRBase: the microRNA database, Release 22.1 (from Griffiths-Jones laboratory) Available from: <a target="_blank" href="https://www.mirbase.org">https://www.mirbase.org</a> (2022)</li><li>. Dharmacon CRISPR Design Tool (from Dharmacon) Available from: <a target="_blank" href="https://www.horizondiscovery.com/en/ordering-and-calculation-tools/crispr-dna-region-designer">https://www.horizondiscovery.com/en/ordering-and-calculation-tools/crispr-dna-region-designer</a> (2022)</li><li>. Off-Spotter (Venetia Pliatsika &amp; Isidore Rigoutsos of the Jefferson Computational Medicine Center) Available from: <a target="_blank" href="https://cm.jefferson.edu/Off-Spotter/">https://cm.jefferson.edu/Off-Spotter/</a> (2022)</li><li>Bao, X. R., Pan, Y., Lee, C. M., Davis, T. H., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+experimental+and+computational+analyses+of+off-target+editing+by+programmable+nucleases.">Tools for experimental and computational analyses of off-target editing by programmable nucleases.</a> Nature Protocols. 16 (1), 10-26 (2021).</li><li>. Dharmacon DharmaFECT Transfection Reagent Cell Type Guide. Choose Dharmacon DharmaFECT 1, 2, 3, or 4 for optimal transfection of siRNA or miRNA reagents Available from: <a target="_blank" href="https://horizondiscovery.com/-/media-Files/Horizon/resources/Selection-guides/dharmafect-cell-type-guidelines.pdf">https://horizondiscovery.com/-/media-Files/Horizon/resources/Selection-guides/dharmafect-cell-type-guidelines.pdf</a> (2022)</li><li>Baffoe-Bonnie, A. B., 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+major+locus+for+hereditary+prostate+cancer+in+Finland:+localization+by+linkage+disequilibrium+of+a+haplotype+in+the+HPCX+region.">A major locus for hereditary prostate cancer in Finland: localization by linkage disequilibrium of a haplotype in the HPCX region.</a> Human Genetics. 117 (4), 307-316 (2005).</li><li>Zhang, R., Peng, Y., Wang, W., Su, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+an+X-linked+microRNA+cluster+in+primates.">Rapid evolution of an X-linked microRNA cluster in primates.</a> Genome Research. 17 (5), 612-617 (2007).</li><li>Ohnuki, Y., Marnell, M. M., Babcock, M. S., Lechner, J. F., Kaighn, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+analysis+of+human+prostatic+adenocarcinoma+cell+lines.">Chromosomal analysis of human prostatic adenocarcinoma cell lines.</a> Cancer Research. 40 (3), 524-534 (1980).</li><li>Beheshti, B., Karaskova, J., Park, P. C., Squire, J. A., Beatty, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+high+frequency+of+chromosomal+rearrangements+in+the+centromeric+regions+of+prostate+cancer+cell+lines+by+sequential+giemsa+banding+and+spectral+karyotyping.">Identification of a high frequency of chromosomal rearrangements in the centromeric regions of prostate cancer cell lines by sequential giemsa banding and spectral karyotyping.</a> Molecular Diagnosis. 5 (1), 23-32 (2000).</li></ol>

यह सीआरआईएसपीआर जीन संपादन प्रक्रिया अन्वेषक को अद्वितीय एमआईआरएनए क्लस्टर विलोपन संयोजनों को ले जाने वाली सेल लाइनों के पूरे पैनल को जल्दी से उत्पन्न करने की अनुमति देती है। इस प्रोटोकॉल में सिंथे?...

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL PCR tubes, flat cap</td>
			<td>Fisher</td>
			<td>14-230-225</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Seal-Rite</td>
			<td>1615-5500</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Corning Costar</td>
			<td>&#160;09761146</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>5x Phusion HF Buffer</td>
			<td>Thermo Scientific</td>
			<td>F-518L</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Fisher</td>
			<td>FB012927</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>08-772-2C</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>Anti-CRISPR-Cas9 antibody [7A9-3A3], Mouse monoclonal</td>
			<td>AbCam</td>
			<td>ab191468</td>
			<td>Western blot reagent; 160 kDa; dilution 1:200</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td>Tisuue culture reagent</td>
		</tr>
		<tr>
			<td>BLAST nucleotide search engine</td>
			<td>National Center for Biotechnology Information</td>
			<td>NA</td>
			<td>Freeware; website: blast.ncbi.nlm.nih.gov</td>
		</tr>
		<tr>
			<td>Blasticidin S, Hydrochloride, Streptomyces griseochromogenes</td>
			<td>Calbiochem</td>
			<td>CAS 589205</td>
			<td>CRISPR reagent; Chemical, Working stock = 1 mg/mL in water</td>
		</tr>
		<tr>
			<td>Countess 3 Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>A50298</td>
			<td>Equipment; Cell counter</td>
		</tr>
		<tr>
			<td>Cryogenic tubes</td>
			<td>Thermo Scientific</td>
			<td>50001012</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>Dharmacon CRISPR Design Tool</td>
			<td>Horizon Discovery Ltd.</td>
			<td>NA</td>
			<td>Freeware; website: horizondiscovery.com/en/ordering-and-calculation-tools/crispr-dna-region-designer</td>
		</tr>
		<tr>
			<td>DharmaFECT siRNA Transfection Reagent #2</td>
			<td>Dharmacon, Inc.</td>
			<td>T-2002-02</td>
			<td>Transfection reagent; LNCaP and PC3-ML cell lines</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher</td>
			<td>&#160;BP231100</td>
			<td>Tisuue culture reagent</td>
		</tr>
		<tr>
			<td>DMEM Medium with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td>
			<td>Corning</td>
			<td>MT10013CV</td>
			<td>Tisuue culture reagent; Media for PC3-ML cells</td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride, Ready Made Solution</td>
			<td>Sigma-Aldrich</td>
			<td>D3072-1ML</td>
			<td>CRISPR reagent; Chemical, Working stock = 1 mg/mL stock in water</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td>Tisuue culture reagent</td>
		</tr>
		<tr>
			<td>Edit-R CRISPR-Cas9 Synthetic tracrRNA, 20 nmol, designed</td>
			<td>Dharmacon, Inc.</td>
			<td>U-002005-20</td>
			<td>CRISPR reagent; Universal tracrRNA oligonucleotides</td>
		</tr>
		<tr>
			<td>Edit-R Modified Synthetic crRNA, 
			desalted/deprotected, 2 nmol</td>
			<td>Dharmacon, Inc.</td>
			<td>crRNA-460XXX</td>
			<td>CRISPR reagent; Designed crRNA oligonucleotides</td>
		</tr>
		<tr>
			<td>EditR Inducible Lentiviral hEF1aBlastCas9 Nuclease Particles, 50 &#956;L, 107 TU/mL</td>
			<td>Dharmacon, Inc.</td>
			<td>VCAS11227</td>
			<td>CRISPR reagent; Lenti-iCas9; Doxycycline-inducible lentiviral Streptococcus pyogenes Cas9 vector system</td>
		</tr>
		<tr>
			<td>Ensembl genomic viewer</td>
			<td>Ensembl</td>
			<td>NA</td>
			<td>Freeware; website: [ensembl.org] Use: Genome browser to identify a nucleotide seuqnces containing the miRNA cluster and surrounding gene/regulatory sequences.</td>
		</tr>
		<tr>
			<td>GAPDH Antibody (FL-335), rabbit polyclonal</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc25778</td>
			<td>Western blot reagent; 37 kDa; dilution 1:500</td>
		</tr>
		<tr>
			<td>Gel/PCR DNA Fragment Extraction Kit</td>
			<td>IBI Scientific</td>
			<td>IB47010</td>
			<td>Nucleic acid gel electrophoresis reagent</td>
		</tr>
		<tr>
			<td>Gibco Fetal Bovine Serum, certified</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>Tisuue culture reagent</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>MilliporeSigma</td>
			<td>H9268</td>
			<td>CRISPR reagent; Chemical, Working stock = 0.8 mg/mL</td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF Membrane</td>
			<td>MilliporeSigma</td>
			<td>IPFL10100</td>
			<td>Western blot reagent; nitrocellulose</td>
		</tr>
		<tr>
			<td>Intercept (TBS) Blocking Buffer</td>
			<td>LI-COR</td>
			<td>927-60001</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td>
			<td>LI-COR</td>
			<td>926-68070</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td>
			<td>LI-COR</td>
			<td>926-32211</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>&#160;5425 R</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>miRBase microRNA viewer</td>
			<td>miRBase</td>
			<td>NA</td>
			<td>Freeware; website: [mirbase.org] Use: Borowser lists all annotated miRNA hairpins, mature miRNA sequences and associated clustered miRNAs mapping within 10 kb.</td>
		</tr>
		<tr>
			<td>NuPAGE 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well</td>
			<td>Invitrogen</td>
			<td>NP0321BOX</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>Odyssey CLx Imaging System</td>
			<td>LI-COR</td>
			<td>CLx</td>
			<td>Equipment; Western blot imaging</td>
		</tr>
		<tr>
			<td>Opti-MEM Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985062</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Owl D2 Wide-Gel Electrophoresis System</td>
			<td>Owl</td>
			<td>D2-BP</td>
			<td>Equipment; Nucleic acid gel electrophoresis system</td>
		</tr>
		<tr>
			<td>PCR Primers, designed (single-stranded DNA oligonucleotides)</td>
			<td>Integrated DNA Technology</td>
			<td>NA</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase (2 U/&#181;L)</td>
			<td>Thermo Scientific</td>
			<td>F530S</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>RIPA Lysis Buffer 10x</td>
			<td>MilliporeSigma</td>
			<td>20-188</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>Proteinase K Solution (20 mg/mL), RNA grade</td>
			<td>Invitrogen</td>
			<td>25530049</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>RNase A, DNase and protease-free (10 mg/mL)</td>
			<td>Thermo Scientific</td>
			<td>EN0531</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>MT10041CV</td>
			<td>Tisuue culture reagent; Media for LNCaP cells</td>
		</tr>
		<tr>
			<td>SnapGene Viewer</td>
			<td>Snap Gene</td>
			<td>NA</td>
			<td>Freeware; website: [snapgene.com]&#160;Use: DNA sequence annotation software program to create a DNA file for gRNA design and which&#160; highlights the miRNA cluster locus (intergenic, intronic), each individual miRNA hairpin sequence belonging to the miRNA cluster, and other nearby coding and non-coding genes and/or regulatory features</td>
		</tr>
		<tr>
			<td>TrypLE Select Enzyme (1x), no phenol red</td>
			<td>Gibco</td>
			<td>12563029</td>
			<td>Tisuue culture reagent; Recombinant trypsin</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>16500100</td>
			<td>Nucleic acid gel electrophoresis reagent</td>
		</tr>
		<tr>
			<td>Veriti 96-Well Fast Thermal Cycler</td>
			<td>ThermoFisher Scientific</td>
			<td>4375305</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>XCell SureLock Mini-Cell Electrophoresis System</td>
			<td>Invitrogen</td>
			<td>EI0001</td>
			<td>Equipment; Protein gel electrophoresis system</td>
		</tr>
	</tbody>
</table>

crispr gene editing tool for microrna cluster network analysis

माइक्रोआरएनए (एमआईआरएनए) कैंसर और मेटास्टेसिस के महत्वपूर्ण सेलुलर नियामकों (ट्यूमर सप्रेसर, प्रो-ऑन्कोजेनिक कारक) के रूप में उभरे हैं। अधिकांश प्रकाशित अध्ययन कैंसर में छोटे आरएनए की भूमिका की विशेषता के दौरान एक एकल एमआईआरएनए पर ध्यान केंद्रित करते हैं। हालांकि, मानव एमआईआरएनए जीन का ~ 30% क्लस्टर इकाइयों में व्यवस्थित किया जाता है जो अक्सर सह-व्यक्त होते हैं, जो नॉनकोडिंग आरएनए विनियमन की एक जटिल और समन्वित प्रणाली का संकेत देते हैं। क्लिनिक में नॉनकोडिंग छोटे आरएनए का अनुवाद करने से पहले ट्यूमर के विकास, कैंसर की आक्रामकता और दवा प्रतिरोध को विनियमित करने के लिए क्लस्टर एमआईआरएनए नेटवर्क सहकारी रूप से कैसे कार्य करते हैं, इसका एक स्पष्ट अंडरटेलिंग आवश्यक है।
प्रोस्टेट कैंसर के संदर्भ में ~ 35,000 बीपी लंबाई में फैले स्थान के भीतर स्थित सात एमआईआरएनए जीन के जीनोमिक क्लस्टर की ऑन्कोजेनिक भूमिका का अध्ययन करने के लिए एक उच्च-थ्रूपुट क्लस्टर नियमित रूप से इंटरस्पेस्ड शॉर्ट पैलिंड्रोमिक रिपीट (सीआरआईएसपीआर) -मध्यस्थता जीन संपादन प्रक्रिया का उपयोग नियोजित किया गया है। इस दृष्टिकोण के लिए, मानव कैंसर सेल लाइनों को 48 घंटे के लिए डॉक्स युक्त माध्यम में उगाए गए डॉक्सीसाइक्लिन (डॉक्स) -इंड्यूसिबल कैस 9 न्यूक्लियस के लिए एक लेंटीवायरस वेक्टर से संक्रमित किया गया था। कोशिकाओं को बाद में सिंथेटिक ट्रांस-एक्टिवेटिंग सीआरआईएसपीआर आरएनए (टीआरएसआरएनए) के साथ सह-संक्रमित किया गया था, जो जीनोमिक साइट-विशिष्ट सीआरआईएसपीआर आरएनए (सीआरआरएनए) ऑलिगोन्यूक्लियोटाइड्स के साथ जटिल था ताकि कैंसर सेल लाइनों की तेजी से पीढ़ी को एक ही प्रयोग के भीतर पूरे एमआईआरएनए क्लस्टर विलोपन और व्यक्तिगत या संयोजन एमआईआरएनए जीन क्लस्टर विलोपन की अनुमति मिल सके।
इस उच्च थ्रूपुट जीन संपादन प्रणाली के फायदे समय लेने वाली डीएनए वेक्टर सबक्लोनिंग से बचने की क्षमता, 24-अच्छी तरह से प्रारूप में अद्वितीय गाइड आरएनए संयोजनों के साथ ट्रांसफेक्टिंग कोशिकाओं में लचीलापन, और क्रूड सेल लाइसेट्स का उपयोग करके कम लागत वाले पीसीआर जीनोटाइपिंग हैं। इस सुव्यवस्थित दृष्टिकोण का उपयोग करने वाले अध्ययन एमआईआरएनए क्लस्टर सदस्यों के बीच कार्यात्मक अतिरेक और सहक्रियात्मक / विरोधी बातचीत को उजागर करने का वादा करते हैं, जो मानव रोग में शामिल जटिल छोटे नॉनकोडिंग आरएनए नेटवर्क को चिह्नित करने में सहायता करेगा और भविष्य के चिकित्सीय डिजाइन को बेहतर ढंग से सूचित करेगा।

इस उच्च थ्रूपुट सीआरआईएसपीआर विलोपन प्रोटोकॉल को एमआईआर -888 क्लस्टर को लक्षित करने वाले सिंथेटिक ऑलिगोन्यूक्लियोटाइड गाइड आरएनए के साथ कैस 9-इंड्यूसिबल एलएनसीएपी और पीसी 3-एमएल मानव कैंसर सेल लाइनों ?...

Watch this Scientific Journal Video about CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis at JoVE.com

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

यह प्रोटोकॉल माइक्रोआरएनए क्लस्टर नेटवर्क विश्लेषण के लिए एक उच्च-थ्रूपुट क्लस्टर नियमित रूप से इंटरस्पेस्ड शॉर्ट पैलिंड्रोमिक रिपीट (सीआरआईएसपीआर) जीन संपादन वर्कफ़्लो का वर्णन करता है जो आनुवंशिक रूप से संशोधित सेल लाइनों के एक पैनल की तेजी से पीढ़ी की अनुमति देता है जो अद्वितीय एमआईआरएनए क्लस्टर सदस्य विलोपन संयोजनों को एक प्रयोग के भीतर 35 केबी जितना बड़ा होता है।

माइक्रोआरएनए क्लस्टर नेटवर्क विश्लेषण के लिए सीआरआईएसपीआर जीन संपादन उपकरण

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

crispr-gene-editing-tool-for-microrna-cluster-network-analysis

Research

JoVE Journal

Biology

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

माइक्रोएरे विश्लेषण के लिए Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

SECM गिरफ्तार अनुक्रम एक ribosome बाध्य polypeptides पृथक उपकरण के रूप में का उपयोग

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

भोजी के विश्लेषण में<em&gt; पेनिसिलियम chrysogenum</em&gt; प्रतिदीप्ति माइक्रोस्कोपी के साथ संयोजन में भुखमरी पैड का उपयोग करके

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

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.

स्पार्क को सिलिंग: कमजोर इलेक्ट्रिक मछली में CRISPR/Cas9 जीनोम संपादन

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

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

नासोनिया विट्रिपेनिस में आरएनएआई और CRISPR जीन संपादन के लिए पुपल और वयस्क इंजेक्शन

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&amp;#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

टीजीएफ-β-मध्यस्थता एंडोथेलियल से मेसेंचिमल ट्रांजिशन (एंडएमटी) और CRISPR/Cas9 जीन संपादन का उपयोग करके एंडएमटी प्रभावकर्ताओं का कार्यात्मक मूल्यांकन

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

इन विट्रो E3 सर्वव्यापकिन लिगाज़ समारोह का विश्लेषण

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

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.

कैस 9 जीन थेरेपी अनुप्रयोगों के लिए हेमटोपोइएटिक स्टेम और पूर्वज कोशिकाओं का जीन संपादन

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

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

ब्लैक-लेग्ड टिक में जीन संपादन के लिए भ्रूण इंजेक्शन तकनीक, इक्सोड्स स्कैपुलारिस

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly 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 worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

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

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

ओरिएंटल फ्रूट फ्लाई बैक्ट्रोसेरा डोरसेलिस के CRISPR/Cas9 म्यूटेनेसिस के लिए प्रोटोकॉल

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 ...