Name: in vivo crispr/cas9 screening to simultaneously evaluate gene function in mouse skin and oral cavity
Uploaded: 2020-11-02T13:00:00
Description: Watch this Scientific Journal Video about In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity at JoVE.com

Dette arbejde blev støttet af et projekt tilskud fra canadian Institute of Health Research (CIHR 365252), Krembil Foundation og Ontario Research Fund Research Excellence Round 8 (RE08-065). Sampath Kumar Loganathan er modtager af en canadisk Cancer Society stipendium (BC-F-16 # 31919).

Denne protokol blev godkendt og udført i overensstemmelse med IACUC fra University of Toronto.
1. Design og kloning af sammenlagte CRISPR-biblioteker
<ol><li>Vælg 4-5 sgRNA'er, der er målrettet mod musegener af interesse fra ressourcer, f.https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design (Broad Institute sgRNA designer) eller CHOPCHOP-server (https://chopchop.cbu.uib.no). Vælg et lige antal ikke-målretnings-sgRNA'er fra f.eks.</li>
	<li>Me...

<ol>
	<li>Beronja, S., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-mediated+gene+function+analysis+in+skin.">RNAi-mediated gene function analysis in skin.</a> Methods in Molecular Biology. 961, 351-361 (2013).</li><li>Beronja, S., Livshits, G., Williams, S., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+functional+dissection+of+genetic+networks+via+tissue-specific+transduction+and+RNAi+in+mouse+embryos.">Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos.</a> Nature Medicine. 16, 821-827 (2010).</li><li>Shalem, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+CRISPR-Cas9+knockout+screening+in+human+cells.">Genome-scale CRISPR-Cas9 knockout screening in human cells.</a> Science. 343, 84-87 (2014).</li><li>Beronja, 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=RNAi+screens+in+mice+identify+physiological+regulators+of+oncogenic+growth.">RNAi screens in mice identify physiological regulators of oncogenic growth.</a> Nature. 501, 185-190 (2013).</li><li>Loganathan, S. 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=Rare+driver+mutations+in+head+and+neck+squamous+cell+carcinomas+converge+on+NOTCH+signaling.">Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling.</a> Science. 367, 1264-1269 (2020).</li><li>Leemans, C. R., Snijders, P. J. F., Brakenhoff, 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=The+molecular+landscape+of+head+and+neck+cancer.">The molecular landscape of head and neck cancer.</a> Nature Reviews Cancer. 18, 269-282 (2018).</li><li>Green, A. C., Olsen, 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=Cutaneous+squamous+cell+carcinoma:+an+epidemiological+review.">Cutaneous squamous cell carcinoma: an epidemiological review.</a> British Journal of Dermatology. 177, 373-381 (2017).</li><li>Campbell, J. 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=pathway+network,+and+immunologic+features+distinguishing+squamous+carcinomas.">pathway network, and immunologic features distinguishing squamous carcinomas.</a> Cell Reports. 23, 194-212 (2018).</li><li>Sanjana, N. E., Shalem, O., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+and+genome-wide+libraries+for+CRISPR+screening.">Improved vectors and genome-wide libraries for CRISPR screening.</a> Nature Methods. 11, 783-784 (2014).</li><li>Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z., Gijsbers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+lentiviral+vector+titration+methods.">Comparison of lentiviral vector titration methods.</a> BMC Biotechnology. 6, 34 (2006).</li><li>Schramek, 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=Direct+in+vivo+RNAi+screen+unveils+myosin+IIa+as+a+tumor+suppressor+of+squamous+cell+carcinomas.">Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor of squamous cell carcinomas.</a> Science. 343, 309-313 (2014).</li><li>Platt, R. 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=CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling.">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159, 440-455 (2014).</li><li>Langmead, B., Trapnell, C., Pop, M., 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=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biology. 10, 25 (2009).</li><li>Li, 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=MAGeCK+enables+robust+identification+of+essential+genes+from+genome-scale+CRISPR/Cas9+knockout+screens.">MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.</a> Genome Biology. 15, 554 (2014).</li><li>Winters, I. P., Murray, C. W., Winslow, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+quantitative+and+multiplexed+in+vivo+functional+cancer+genomics.">Towards quantitative and multiplexed in vivo functional cancer genomics.</a> Nature Reviews Genetics. 19, 741-755 (2018).</li><li>Rogers, Z. N., 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=Mapping+the+in+vivo+fitness+landscape+of+lung+adenocarcinoma+tumor+suppression+in+mice.">Mapping the in vivo fitness landscape of lung adenocarcinoma tumor suppression in mice.</a> Nature Genetics. 50, 483-486 (2018).</li><li>Chow, R. 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=AAV-mediated+direct+in+vivo+CRISPR+screen+identifies+functional+suppressors+in+glioblastoma.">AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma.</a> Nature Neuroscience. 20, 1329-1341 (2017).</li><li>Wang, G., 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=Mapping+a+functional+cancer+genome+atlas+of+tumor+suppressors+in+mouse+liver+using+AAV-CRISPR&#8211;mediated+direct+in+vivo+screening.">Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR&#8211;mediated direct in vivo screening.</a> Science Advances. 4, 5508 (2018).</li><li>Chan, K., Tong, A. H. Y., Brown, K. R., Mero, P., Moffat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pooled+CRISPR-based+genetic+screens+in+mammalian+cells.">Pooled CRISPR-based genetic screens in mammalian cells.</a> Journal of Visualized Experiments. (151), e59780 (2019).</li><li>Hart, 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=High-resolution+CRISPR+screens+reveal+fitness+genes+and+genotype-specific+cancer+liabilities.">High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities.</a> Cell. 163, 1515-1526 (2015).</li></ol>

CRISPR/Cas9 genomredigering har været meget udbredt i in vitro- og in vivo-undersøgelser til at undersøge genfunktioner og cellulære processer. De fleste in vivo-undersøgelser anvender CRISPR/Cas9-genredigerede celler, der er podet ind i en dyremodel (allograft eller xenograft). Selv om dette er et kraftfuldt værktøj til at studere kræft genetik og cellulære funktioner, det stadig mangler den indfødte væv mikromiljø og kan fremkalde sårende og / eller immunrespons.
For at overvind...

En af udfordringerne for kræftforskningen i den postgenomiske æra er at udvinde den enorme mængde genomdata for kausale genmutationer og identificere knuder i gennetværket, der kan målrettes terapeutisk. Selv om bioinformatiske analyser har bidraget enormt til at nå disse mål, er det en forudsætning at etablere effektive in vitro- og in vivo-modeller for at dechifrere kompleksiteten af biologiske systemer og sygdomstilstande og for at muliggøre udvikling af lægemidler. Mens konventionelle transgene musemodeller er blevet brugt i vid udstrækning til in vivo kræft genetik undersøgelser, deres omkostninger-, tid-og arbejdskrævende karakter har stort set forbudt systematisk analyse af de hundredvis af formodede kræft gener optrevlet af moderne genomik. For at overvinde denne flaskehals kombinerede vi en tidligere etableret ultralydsstyret i livmoderinjektionsmetode1,2 med en CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) genredigeringsteknologi3 for samtidig at fremkalde og studere funktionstabsmutationer af hundredvis af gener i hud og mundhule hos en enkelt mus.
Den metode, der er beskrevet her, bruger ultralydsstyrede injektioner af manipulerede lentivirus i fosterhulen af levende museembryoner på embryonal dag E9.5. Den lentivirale last, der indeholder CRISPR/Cas9-komponenterne, transduce den enkeltlags overflade ectoderm, som senere giver anledning til epitel af huden og mundhulen. Huden er sammensat af en ydre epidermis, efterfulgt af kældermembran og dermis. Epidermis er et stratificeret epitel bestående af et indre, basalt lag, som opretholder kontakt med kældermembranen og har proliferativ og stamcellekapacitet. Det basale lag giver anledning til de differentierede lag ovenfor, såsom spinøse, granulære og stratum corneum lag2,4. Undersøgelser af afstamningssporing viser, at denne direkte in vivo CRISPR/Cas9-metode genmanipulerer vævsbeboende stamceller i det basale lag, der fortsætter i hele voksenalderen. Da lentivirus kan titreres for at transduce E9.5 overflade ectoderm ved klonisk tæthed, kan denne metode bruges til at generere mosaikmus, der huser tusinder af diskrete gen knock-out kloner. Næste generations sekventering kan derefter bruges til at analysere effekten af CRISPR/Cas9-medieret genabsering inden for disse kloner på en multiplekseret måde5.
Vi har for nylig brugt denne metode til at vurdere funktionen af 484 gener, der viser tilbagevendende mutationer i humane hoved og hals Squamous Cell Carcinoma (HNSCC)5. HNSCC er en ødelæggende kræft med en høj dødelighed på 40-50%, og det er den 6th mest almindelige kræft på verdensplan6. HNSCC opstår i slimhindeforinger af de øvre luftveje eller mundhulen og er forbundet med tobaks- og alkoholforbrug eller human papillomavirus (HPV) infektion. Kutan squamous Cell Carcinoma (SCC) er hudtumorer og repræsenterer den næsthyppigste kræftform hos mennesker7. Kutan SCC og HNSCC er histologisk og molekylært meget ens, med en høj procentdel af tilfælde udviser ændring i TP53, PIK3CA, NOTCH1, og HRAS8. Mens der kun er en håndfuld gener muteret ved høj frekvens, er der hundredvis af gener, der findes muteret ved lav frekvens (< 5%), et fænomen, der almindeligvis omtales som langtidshalefordelingen. Da størstedelen af langhalegenerene mangler biologisk eller klinisk validering, brugte vi denne in vivo CRISPR-screeningsteknologi til at modellere tab af funktion af disse gener i tumor-tilbøjelige mus med sensibiliserende mutationer i p53, Pik3ca eller Hras og identificerede flere nye tumorundertrykkergener, der samarbejder med p53, Pik3ca eller Hras for at udløse tumorudvikling5.
Her beskriver vi en detaljeret protokol til generering af multiplexed sgRNA lentivirale CRISPR sgRNA-biblioteker og udfører CRISPR/Cas9-gensmittende skærme i museoverfladens ectoderm. Bemærk, at denne metode kan tilpasses til at inkorporere andre genmanipulationsteknologier såsom CRISPR-aktivering (CRISPRa) og CRISPR-inaktivering (CRISPRi) eller ændres til at målrette andre organsystemer med musen for at studere genfunktioner.

<table>
	<tbody>
		<tr>
			<td>0.45 micron filter</td>
			<td>Sigma</td>
			<td>S2HVU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>12k or 92k oligo chip</td>
			<td>Customarray Inc. (Genscript)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm cell culture plates</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>293FT</td>
			<td>Invitrogen</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>293NT</td>
			<td>Systems Biosciences</td>
			<td>LV900A-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase</td>
			<td>NEB</td>
			<td>M0290L</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>NEB</td>
			<td>9804S</td>
			<td></td>
		</tr>
		<tr>
			<td>BsmBI</td>
			<td>NEB</td>
			<td>R0580L</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromic gut suture</td>
			<td>Covidien</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deep sequencing (Next-Seq or Hi-Seq)</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA-cleanup kit</td>
			<td>Zymo Research</td>
			<td>D4008</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAesy Blood and Tissue DNA extraction kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr>
			<td>Endura electrocompetent cells</td>
			<td>Lucigen</td>
			<td>60242-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>Drummond</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Speed Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>MLS-50</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Wisent Technologies</td>
			<td>800-011-LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-prep plasmid Kit</td>
			<td>Frogga Bio</td>
			<td>PDH300</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse oxygen anaesthesia system</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II micromanipulator</td>
			<td>Drummond</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 3.1 (Buffer for BsmBI)</td>
			<td>NEB</td>
			<td>R0580L</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle sharpener</td>
			<td>Sutter Instrument</td>
			<td>BV-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo cleanup kit</td>
			<td>Zymo research</td>
			<td>D4060</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE purified illumina sequencing primer</td>
			<td>IDT DNA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEI (polyethyleneimine)</td>
			<td>Sigma</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Permoplast modeling clay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petridish with central opening</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 Polymerase 2x Master mix</td>
			<td>NEB</td>
			<td>M0494L</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Fluorometric Quantification</td>
			<td>Invitrogen</td>
			<td>Q33327</td>
			<td></td>
		</tr>
		<tr>
			<td>Semicircular Silicone plug</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone membrane</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo2000 ultrasound system</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo crispr/cas9 screening to simultaneously evaluate gene function in mouse skin and oral cavity

Genetisk modificerede musemodeller (GEMM) har været medvirkende til at vurdere genfunktion, modellere menneskelige sygdomme og fungere som præklinisk model til vurdering af terapeutiske veje. Men deres tids-, arbejds- og omkostningsintensive natur begrænser deres nytteværdi for systematisk analyse af genfunktion. De seneste fremskridt inden for genomredigeringsteknologier overvinder disse begrænsninger og giver mulighed for hurtig generering af specifikke genforbræte direkte inden for specifikke museorganer på en multiplekseret og hurtig måde. Her beskriver vi en CRISPR/Cas9-baseret metode (Clustered Regularly Interspaced Short Palindromic Repeats) til at generere tusindvis af gen knock-out kloner i epitel af huden og mundhulen af mus, og give en protokol beskriver de nødvendige skridt til at udføre en direkte in vivo   CRISPR skærm for tumor suppressor gener. Denne tilgang kan anvendes på andre organer eller andre CRISPR/Cas9-teknologier såsom CRISPR-aktivering eller CRISPR-inaktivering for at studere genernes biologiske funktion under vævshomøostase eller i forskellige sygdomsindstillinger.

Figur 1A viser designet af oligonukleotider til multiplexing af flere brugerdefinerede CRISPR-biblioteker på en omkostningseffektiv måde på en enkelt 12k eller 92k oligochip. Når sgRNA'erne (blå farvekodet) er valgt, er oligonukleotider designet med begrænsningssteder (orangefarvet BsmBI) og biblioteksspecifikke PCR primer-par (grøn farvekodet). Flere biblioteker kan designes ved hjælp af unik kombination af primer par til multiplexing i en enkelt oligo chip. Når PCR forstærke bibl...

Watch this Scientific Journal Video about In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity at JoVE.com

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Her beskriver vi en hurtig og direkte in vivo CRISPR/Cas9 screeningsmetode ved hjælp af ultralydstyret i livmoderembryo-lentivirale injektioner for samtidig at vurdere funktioner af flere gener i huden og mundhulen hos immunkontromerende mus.

I Vivo CRISPR/Cas9 Screening til samtidig evaluering af genfunktion i musehud og mundhule

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

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Udvælgelsesafhængig og uafhængig generering af CRISPR / Cas9-medierede Gene Knockouts i Pattedyrceller

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

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En standardmetode til at undersøge ejendommen mutagenicitet som en funktion af punkt Mutation reparation katalyseret af CRISPR/Cas9 og SsODN i humane celler

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

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

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

Mikroinjektion af CRISPR/Cas9 Protein i kanal havkat, Ictalurus punctatus, embryoner til Gene Editing

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

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-medieret målrettede Integration In Vivo ved hjælp af en homologi-medieret End at deltage-baseret strategi

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Effektiv produktion og identifikation af CRISPR/Cas9-genereret gen Knockouts i Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Højeffektive gen afbrydelse af Murine og menneskelige hæmatopoietisk stamceller ved CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

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

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

CRISPR/Cas9 gen redigering at gøre betinget mutanter af menneskelige Malaria parasitten, P. falciparum

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

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

CRISPR/Cas9 ribonucleoprotein-medieret præcis Genredigering ved Rørelektroporation

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