Name: selection-dependent and independent generation of crispr/cas9-mediated gene knockouts in mammalian cells
Uploaded: 2017-06-16T15:00:00
Description: Watch this Scientific Journal Video about Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells at JoVE.com

Os autores gostariam de reconhecer Gissell Sanchez, Megan Lee e Jason Estep para assistência experimental, e Weifeng Gu e Xuemei Chen por compartilhar reagentes.

1. Identificação de regiões de homologia em torno da eliminação desejada NOTA: necessário somente se estiver usando a edição baseada em seleção. <ol><li> Selecione duas regiões, inicialmente 1,5-2 kb, em ambos os lados do locus de exclusão desejado, que servirá como braços de homologia no modelo HDR ( Figura 1A ). Identifique as regiões que carecem de sites de reconhecimento BsaI em ambos os lados (GGTCTC) para facilitar a clonagem. Se os sítio...

<ol>
	<li>Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+with+zinc-finger+nucleases.">Genome engineering with zinc-finger nucleases.</a> Genetics. 188 (4), 773-782 (2011).</li><li>Kim, Y. G., Cha, J., Chandrasegaran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+restriction+enzymes:+zinc+finger+fusions+to+Fok+I+cleavage+domain.">Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.</a> Proc Natl Acad Sci U S A. 93 (3), 1156-1160 (1996).</li><li>Bibikova, 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=Stimulation+of+homologous+recombination+through+targeted+cleavage+by+chimeric+nucleases.">Stimulation of homologous recombination through targeted cleavage by chimeric nucleases.</a> Mol Cell Biol. 21 (1), 289-297 (2001).</li><li>Bibikova, M., Golic, M., Golic, K. G., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+chromosomal+cleavage+and+mutagenesis+in+Drosophila+using+zinc-finger+nucleases.">Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.</a> Genetics. 161 (3), 1169-1175 (2002).</li><li>Porteus, M. H., Cathomen, T., Weitzman, M. D., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+targeting+mediated+by+adeno-associated+virus+and+DNA+double-strand+breaks.">Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks.</a> Mol Cell Biol. 23 (10), 3558-3565 (2003).</li><li>Bogdanove, A. J., Voytas, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAL+effectors:+customizable+proteins+for+DNA+targeting.">TAL effectors: customizable proteins for DNA targeting.</a> Science. 333 (6051), 1843-1846 (2011).</li><li>Miller, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+TALE+nuclease+architecture+for+efficient+genome+editing.">A TALE nuclease architecture for efficient genome editing.</a> Nat Biotechnol. 29 (2), 143-148 (2011).</li><li>Moscou, M. J., Bogdanove, 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=A+simple+cipher+governs+DNA+recognition+by+TAL+effectors.">A simple cipher governs DNA recognition by TAL effectors.</a> Science. 326 (5959), 1501 (2009).</li><li>Christian, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+double-strand+breaks+with+TAL+effector+nucleases.">Targeting DNA double-strand breaks with TAL effector nucleases.</a> Genetics. 186 (2), 757-761 (2010).</li><li>Jiang, W., Marraffini, 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=CRISPR-Cas:+New+Tools+for+Genetic+Manipulations+from+Bacterial+Immunity+Systems.">CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems.</a> Annu Rev Microbiol. 69, 209-228 (2015).</li><li>Barrangou, R., 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+provides+acquired+resistance+against+viruses+in+prokaryotes.">CRISPR provides acquired resistance against viruses in prokaryotes.</a> Science. 315 (5819), 1709-1712 (2007).</li><li>Karginov, F. V., Hannon, G. J. <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+system:+small+RNA-guided+defense+in+bacteria+and+archaea.">The CRISPR system: small RNA-guided defense in bacteria and archaea.</a> Mol Cell. 37 (1), 7-19 (2010).</li><li>Wiedenheft, B., Sternberg, S. H., Doudna, J. A. <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+genetic+silencing+systems+in+bacteria+and+archaea.">RNA-guided genetic silencing systems in bacteria and archaea.</a> Nature. 482 (7385), 331-338 (2012).</li><li>Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J., Soria, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervening+sequences+of+regularly+spaced+prokaryotic+repeats+derive+from+foreign+genetic+elements.">Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.</a> J Mol Evol. 60 (2), 174-182 (2005).</li><li>Pourcel, C., Salvignol, G., Vergnaud, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+elements+in+Yersinia+pestis+acquire+new+repeats+by+preferential+uptake+of+bacteriophage+DNA,+and+provide+additional+tools+for+evolutionary+studies.">CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.</a> Microbiology. 151 (Pt 3), 653-663 (2005).</li><li>Brouns, 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=Small+CRISPR+RNAs+guide+antiviral+defense+in+prokaryotes.">Small CRISPR RNAs guide antiviral defense in prokaryotes.</a> Science. 321 (5891), 960-964 (2008).</li><li>Deltcheva, 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=CRISPR+RNA+maturation+by+trans-encoded+small+RNA+and+host+factor+RNase+III.">CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.</a> Nature. 471 (7340), 602-607 (2011).</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>Marraffini, L. A., Sontheimer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+interference+limits+horizontal+gene+transfer+in+staphylococci+by+targeting+DNA.">CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.</a> Science. 322 (5909), 1843-1845 (2008).</li><li>Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas9-crRNA+ribonucleoprotein+complex+mediates+specific+DNA+cleavage+for+adaptive+immunity+in+bacteria.">Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.</a> Proc Natl Acad Sci U S A. 109 (39), E2579-E2586 (2012).</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>Davis, A. J., Chen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double+strand+break+repair+via+non-homologous+end-joining.">DNA double strand break repair via non-homologous end-joining.</a> Transl Cancer Res. 2 (3), 130-143 (2013).</li><li>Guirouilh-Barbat, 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=Impact+of+the+KU80+pathway+on+NHEJ-induced+genome+rearrangements+in+mammalian+cells.">Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells.</a> Mol Cell. 14 (5), 611-623 (2004).</li><li>Moore, J. K., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+and+genetic+requirements+of+two+pathways+of+nonhomologous+end-joining+repair+of+double-strand+breaks+in+Saccharomyces+cerevisiae.">Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.</a> Mol Cell Biol. 16 (5), 2164-2173 (1996).</li><li>Liang, F., Han, M., Romanienko, P. J., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homology-directed+repair+is+a+major+double-strand+break+repair+pathway+in+mammalian+cells.">Homology-directed repair is a major double-strand break repair pathway in mammalian cells.</a> Proc Natl Acad Sci U S A. 95 (9), 5172-5177 (1998).</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=Highly+specific+and+efficient+CRISPR/Cas9-catalyzed+homology-directed+repair+in+Drosophila.">Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila.</a> Genetics. 196 (4), 961-971 (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>Engler, C., Kandzia, R., Marillonnet, 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+one+pot,+one+step,+precision+cloning+method+with+high+throughput+capability.">A one pot, one step, precision cloning method with high throughput capability.</a> PLoS One. 3 (11), e3647 (2008).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8 (11), 2281-2308 (2013).</li><li>Luo, Y., Lin, L., Bolund, L., Sorensen, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+construction+of+rAAV-based+gene+targeting+vectors+by+Golden+Gate+cloning.">Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning.</a> Biotechniques. 56 (5), 263-268 (2014).</li><li>Green, M. R., Sambrook, J., Sambrook, 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> Molecular cloning: a laboratory manual. , (2012).</li><li>Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+homology-directed+human+genome+engineering+by+controlled+timing+of+CRISPR/Cas9+delivery.">Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.</a> Elife. 3, e04766 (2014).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins.">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Res. 24 (6), 1012-1019 (2014).</li></ol>

O sistema CRISPR / Cas9 permitiu uma geração eficiente de modificações genômicas estáveis, que proporcionam uma alternativa mais consistente a outros métodos de manipulação transitória. Aqui, apresentamos dois métodos para identificação rápida de knockouts de genes CRISPR / Cas9 em linhas celulares de mamíferos. Ambos os métodos requerem pouco material celular, pelo que o teste pode ser feito em estágios iniciais da cultura clonal, economizando tempo e reagentes. Para aumentar a eficiência de ambos os ...

<html> Alterações genéticas estáveis ​​oferecem uma vantagem sobre os métodos transitórios de perturbação celular, que podem ser variáveis ​​em sua eficiência e duração. A edição genômica tornou-se cada vez mais comum nos últimos anos devido ao desenvolvimento de nucleases específicas do alvo, como as nucleases de zinco-dedo 1 , 2 , 3 , 4 , 5 , as nucleases efetoras do tipo ativador da transcrição (TALENs) 6 , 7 , 8 , 9 E nucleases guiadas por RNA derivadas do sistema de repetições palindrômicas curtas agrupadas, regularmente intercaladas (CRISPR) 10 . A maquinaria de edição CRISPR / Cas9 é adaptada de um sistema imunológico que bactérias e archaea usam para se defender contra infecções viraisAss = &quot;xref&quot;&gt; 11 , 12 , 13 . Neste processo, fragmentos curtos de 20-30 nt de sequência viral invasora são incorporados em um locus genômico como &quot;espaçadores&quot; flanqueados por unidades repetitivas 14 , 15 . A transcrição subsequente e o processamento de RNA geram pequenos RNAs 16 (CRRNAs) associados ao CRISPR que, juntamente com um CRRNA 17 (traRRNA) trans-ativador, se reúnem com a endonuclease efectora Cas9. Os crRNAs proporcionam assim especificidade ao alvo de Cas9, guiando o complexo para separar sequências de DNA viral complementares e prevenir novas infecções 18 , 19 . Qualquer sequência de &quot;protospacer&quot; no DNA alvo pode servir como fonte do crRNA, desde que seja diretamente 5 &#39;para um motivo adjacente prótespacer curto (PAM), NGG no caso de S. pyogenes Cas9 <sUp class = &quot;xref&quot;&gt; 20. A ausência da sequência PAM perto do espaçador no locus CRISPR do host distingue-se de si mesmo e não-auto, evitando a segmentação do hospedeiro. Devido à sua universalidade e flexibilidade, este sistema biológico foi poderosamente adaptado para a edição genômica, de modo que quase qualquer site de DNA adjacente ao PAM pode ser direcionado. Nesta versão, uma modificação adicional fundiu o crRNA e o tracrRNA em um único componente guia RNA (sgRNA) que é carregado na proteína Cas9 21 . Após a expressão de Cas9 e um sgRNA em células eucarióticas, a proteína Cas9 corta as duas cadeias de DNA no locus alvo. Na ausência de uma região adequada de sequência homóloga, a célula corrige esta ruptura através de união final não homóloga (NHEJ) 22 , 23 , 24 , que tipicamente introduz pequenas deleções ou, raramente, inserções. Ao segmentar umQuadro de leitura, o reparo provavelmente leva a um quadro de translação que produz um produto proteico não funcional. Em contraste, quando fornecido com um modelo exógeno com grandes regiões de homologia, a célula pode corrigir a ruptura de duas vertentes por reparo direcionado por homologia 25 , 26 . Esta rota permite exclusões, substituições ou inserções precisas maiores no genoma, juntamente com a introdução de marcadores de seleção excisáveis 27 . Aqui, apresentamos protocolos para gerar linhas celulares knockout por qualquer um desses dois métodos CRISPR / Cas9 ( Figura 1A ). A abordagem do NHEJ usa um único site de corte de sgRNA e rastreio independente de seleção e, portanto, requer pouca preparação antecipada. Ao usar este método, os RNAs de guia complementares aos exons próximos ao final 5 &#39;da transcrição, que são mais propensos a produzir um knockout, devem ser projetados. Desde o modificatOs íons para o genoma neste caso são pequenos, a triagem para clones knockout baseia-se em manchas de ponto, onde o produto proteico é avaliado de forma eficiente. Utilizamos a geração de linhas knockout de proteína ELAV-like 1 (ELAVL1) como exemplo. A segunda abordagem baseia-se na reparação direta de homologia (HDR) e usa dois sites de corte de sgRNA que abrangem o gene ou região de interesse, permitindo deleções de dezenas de kb. Um plasmídeo com duas regiões de homologia que flanqueiam os sites de clivagem fornece um modelo de substituição ( Figura 1B ), introduzindo um marcador de resistência selecionável que aumenta a eficiência da geração de nocaute. Este método também pode ser adaptado para introduzir modificações de genes com armas de homologia devidamente projetadas. Neste caso, a integração de um novo fragmento de DNA permite a triagem baseada em PCR ( Figura 1C ). Aqui, usamos a geração de linhas knockout do membro da família Pumilio RNA vinculativo 2 (PUM2) como exemplo.

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">Competent E. coli cells</td>
			<td style="width:95px;"></td>
			<td style="width:120px;"></td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plasmid prep kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pSpCas9(BB) plasmid</td>
			<td>Addgene</td>
			<td>42230</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BbsI enzyme</td>
			<td>ThermoFisher</td>
			<td>FD1014</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T7 DNA ligase</td>
			<td>NEB</td>
			<td>M0318L</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tango Buffer</td>
			<td>ThermoFisher</td>
			<td>BY5</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PlasmidSafe exonuclease</td>
			<td>Epicentre</td>
			<td>E3105K</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Q5 hot start high fidelity polymerase</td>
			<td>NEB</td>
			<td>M0494A</td>
			<td>HA amplification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC19-BsaI</td>
			<td></td>
			<td></td>
			<td>Modified pUC19 plasmid, mutated existing BsaI site and inserted two outward facing BsaI sites after BamHI/EcoRI digestion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGolden-Neo</td>
			<td>Addgene</td>
			<td>51422</td>
			<td>Resistance cassette</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGolden-Hygro</td>
			<td>Addgene</td>
			<td>51423</td>
			<td>Resistance cassette</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BsaI enzyme</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td>Homology arm contruction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T7 DNA ligase</td>
			<td>NEB</td>
			<td>M0318L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PlasmidSafe exonuclease</td>
			<td>Epicentre</td>
			<td>E3105K</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toothpicks</td>
			<td></td>
			<td></td>
			<td>bacterial PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq polymerase</td>
			<td></td>
			<td></td>
			<td>bacterial colony PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293 human cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Corning</td>
			<td>MT35010CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Strep (opt.)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plates</td>
			<td>BioLite</td>
			<td>12556004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TransIT-LTI Transfection Reagent</td>
			<td>Mirus</td>
			<td>MIR2300</td>
			<td>for lipofection only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM</td>
			<td>ThermoFisher</td>
			<td>31985062</td>
			<td>for lipofection only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G418 (Neomycin)</td>
			<td>Sigma Aldrich</td>
			<td>A1720-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hygromycin</td>
			<td>Sigma Aldrich</td>
			<td>H3274-250MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plates</td>
			<td>ThermoFisher</td>
			<td>12556008</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Passive Lysis Buffer, 5x</td>
			<td>Promega</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">1xSDS loading buffer</td>
			<td></td>
			<td></td>
			<td>recipe decribed in protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrocellulose Membrane</td>
			<td>Bio-Rad</td>
			<td>162-0115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TBST</td>
			<td></td>
			<td></td>
			<td>recipe decribed in protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dehydrated milk</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SuperSignal West Dura Extended Duration Substrate</td>
			<td>ThermoFisher</td>
			<td>34075</td>
			<td>for HRP-conjugated secondary antibodies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Extracta DNA prep for PCR</td>
			<td>Quantabio</td>
			<td>95091-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KOD polymerase</td>
			<td>Novagen</td>
			<td>71316</td>
			<td></td>
		</tr>
	</tbody>
</table>

selection-dependent and independent generation of crispr/cas9-mediated gene knockouts in mammalian cells

O sistema de engenharia do genoma CRISPR / Cas9 revolucionou a biologia ao permitir uma edição precisa do genoma com pouco esforço. Guiado por um único guia de RNA (sgRNA) que confere especificidade, a proteína Cas9 cliva ambas as cadeias de DNA no locus alvo. A ruptura do DNA pode desencadear união final não homóloga (NHEJ) ou reparação direcionada a homologia (HDR). O NHEJ pode introduzir pequenas deleções ou inserções que levam a mutações de mudança de quadro, enquanto o HDR permite perturbações maiores e mais precisas. Aqui, apresentamos protocolos para geração de linhas celulares knockout através do acoplamento de métodos CRISPR / Cas9 estabelecidos com duas opções para seleção / rastreio a jusante. A abordagem NHEJ usa um único local de corte de sgRNA e rastreio independente de seleção, onde a produção de proteína é avaliada por imunotransferência ponto de uma maneira de alto rendimento. A abordagem HDR usa dois sites de corte de sgRNA que abrangem o gene de interesse. Juntamente com um modelo de HDR fornecido, esse método pode ser excluídoDe dezenas de kb, auxiliado pelo marcador de resistência selecionado inserido. São discutidas as aplicações e vantagens apropriadas de cada método.

Para a geração de linhas knockout ELAVL1, um anticorpo robusto estava disponível, então a edição usando sgRNAs simples ( Figura 1A , esquerda) foi realizada, seguida de imunotransferência por pontos. Três sgRNAs foram transfectados de forma independente para comparar eficiências e descartar os efeitos alvo nos clones resultantes. Após a coleta e mancha de lisados ​​celulares de populações clonais em duas membranas de nitrocelulose, as manchas...

Watch this Scientific Journal Video about Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells at JoVE.com

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

Os avanços recentes na capacidade de manipular geneticamente as linhas celulares somáticas possuem grande potencial para pesquisas básicas e aplicadas. Aqui, apresentamos duas abordagens para CRISPR / Cas9 gerado produção knockout e triagem em linhas de células de mamíferos, com e sem o uso de marcadores selecionáveis.

Geração independente dependente da seleção e de genes independentes de CRISPR / Cas9 em células de mamíferos

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

The goal of this procedure is to isolate and identify clonal knockout cell lines generated by two types of CRIPSR/Cas9-mediated genome editing. One method detects protein knockout caused by nonhomologous end joining while the other detects genomic perturbations generated by homology-directed repair. This method is particularly useful in the field of molecular biology, where generation of cell lines with genetic perturbations is an important tool in dissecting gene function.The main advantage of this technique is that it is a quick and relatively high-throughput method that can be easily executed using basic molecular biology techniques. Nonhomologous end joining introduces small deletions or insertions, and are assessed by a dot immunoblot, while homology-directed repair allows for larger and more precise perturbations, and are assessed by colony PCR. This experiment uses T-REx-293 cells cultured in DMEM media, supplemented with 10%FBS at 37 degrees Celsius, and 5%carbon dioxide.Prior to transvection, plate the cells onto six well plates and include a well for an untransvected control. Grow the cells to approximately 70%confluency. Transvect the cells with 2.5 micrograms of total plasmid using a commercial transvection protocol.For transvection of cells without selection, use 2.5 micrograms of Cas9-sgRNA plasmid. For transvection of cells with selection, use 0.75 micrograms of Cas9-sgRNA1, 0.75 micrograms of Cas9-sgRNA2, and one microgram of homologous recombination template. Incubate the transvected cells and the untransvected control at 37 degrees Celsius and 5%carbon dioxide for 48 hours.For cells transvected with selection, begin the drug selection by treating the cells with the appropriate drug, which is Neomycin in this case. Return the six-well plate to the incubator. Observe the treated cells once a day to check for rates of cell death, until all cells in the untransvected control die.To isolate clonal populations, first grow the cells to 100%confluency in the original well after selection. Next make serial dilutions of the cells and count the cells. Since the proper generation of monoclonal colonies is key to obtaining a full knockout, care must be taken to determine the correct dilution for seating cells at the desired density.Once the correct dilution has been determined, seat the cells into 96-well plates at a density of 0.33 cells per well. Seating three 96=well plates is a good starting point to ensure isolation of more than one correct clone. Over a two-to-four week period, observe the colonies until colonies are visible to the eye.These representative images show colonies at different stages of growth. A single cell at seating, cells at day five, and a well-established colony at day 10. Using a sterile pipette tip, pick visible colonies with care and precision, and reseat in new wells to encourage monolayer growth.Continue growing the cells at 37 degrees Celsius and 5%carbon dioxide. Knockout candidates without selection are screened by dot immunoblot to assess the protein product in a high-throughput manner. After growing individual clones to 50 to 100%confluency, dislodge the monolayer of cells by pipetting within the well.Aliquot 90 microliters of the 100 microliter total volume from each well to a clean microcentrifuge tube. For the purposes of this video, only two samples will be processed. Spin down at 6, 000 RPM for five minutes, remove the media, and lyse the cell pallet in 10 microliters of 1X SDS loading buffer.Add 90 microliters of new media to the remainder of the cells in the well, and return the plate to the incubator to continue propagating the cells. Pipette one microliter of cell lysate onto a dry nitrocellulose membrane to form a dot. Blot each sample twice on two separate membranes, creating two identical patterns of samples.Block the membranes in 5%milk in TBST at room temperature, with rocking, for one hour. After one hour, blot the membranes with primary antibodies at the recommended primary antibody dilution, and TBST plus 5%milk. On one membrane use the primary antibody against the target protein, ELAVL1 in this experiment.On the other membrane, use the primary antibody for a control protein that is not expected to change, PUM2 is used here. Incubate at room temperature for one hour. Remove the primary antibody solution from each blot, add TBST, and incubate for five minutes.In this manner, wash the blots three times with TBST. Next, add to each membrane the appropriate HRP conjugated secondary antibody, and blot at room temperature for one hour. After one hour, wash the membranes three times with TBST for five minutes each time.Apply a chemiluminescent substrate solution to the blots, following the manufacturer's instructions, and image the blotted membranes on a digital chemiluminescence imager. Lastly, quantify dot intensities for the target and control proteins using the appropriate software, and analyze the results as described in the text protocol. Candidates with the selectable resistance marker are screened by colony PCR.Begin this procedure by duplicating individual colonies in a new 96-well plate, and growing them to 100%confluency for preparation of cell lysates. Remove the media from one set of the clones, resuspend the cells from each well in 30 microliters of extraction buffer from a commercial DNA preparation kit, and transfer to a clean 1.5 milliliter microcentrifuge tube. Heat the solution to 96 degrees Celsius for 15 minutes, and then let cool to room temperature.Add 30 microliters of stabilization buffer to each microcentrifuge tube and mix well. To identify wild-type and monoallelic or biallelic mutant lines by colony PCR, design two separate sets of PCR primers to amplify regions around the homology arms based on either successful or unsuccessful integration of the resistance cassette. On each side, use a common primer that anneals outside of the homology arm.To test for the wild-type allele, use the common primer with the corresponding paired primer that is complementary to the endogenous sequence spanning the homology region. To test for the desired mutation, use the common primer with another paired primer, complementary to the inserted resistance cassette. For each sample to be tested, prepare a reaction mixture containing the following, 0.5 microliters of cell lysate, 1.25 microliters of 10X KOD buffer, 0.75 microliters of 25 millimolar magnesium sulfate, 1.25 microliters of two millimolar dNTPs, 0.375 microliters of forward primer, 0.375 microliters of reverse primer, 0.25 microliters of KOD polymerase, and 7.75 microliters of water.Perform PCR using the following conditions, 95 degrees Celsius for two minutes, followed by 25 cycles of 95 degree Celsius for 20 seconds, primer melting temperature for 10 seconds, and 70 degrees Celsius for 20 seconds per KB.Subsequently, visualize the PCR products by agarose gel electrophoresis. Genome editing using non-homologous end joining was performed to generate ELAVL1 knockout lines followed by dot immunoblot, probing for ELAVL1 and PUM2. Clones that displayed little control signal were marked with an X, and excluded from further analysis.Clones with low ELAVL1 signals were further tested by western blot. Confirmed candidates are indicated in green, and invalidated candidates are indicated in gray. This graphic shows the ratios of ELAVL1 to control protein signal in increasing order.In most cases, clonal populations with the lowest relative ELAVL1 signal were correctly identified as knockouts. Candidates generated by the homology directed repair method were screened by colony PCR, and the PCR products visualized by agarose gel electrophoresis. Shown are example results using primers spanning the upstream integration junction using endogenous inside primers and knockout inside primers.Primers were first validated in both parental cells and bulk selected cells. Results from 24 individual candidate clones are shown together with the expected band patters for wild-type, knockout, and monoallelic deletion clones. The black arrows indicate the correct amplification products, stars indicate biallelic knockout clones.The biallelic mutant lines were further validated by western blot. Nonhomologous end joining uses a single Cas9 cut and selection-independent screening, thus requiring little up-front preparation, and screening for knockout clones by dot blots can quickly determine if a knockout generation was successful. PCR-based screening accurately detects correct candidates with large genomic perturbations generated by homology-directed repair.These techniques enable researchers in molecular biology to easily and efficiently identify knockouts, allowing them to explore normal or dysfunctional cellular processes using cell lines. After watching this video, you should have a good understanding of how to isolate and identify clonal knockout cell lines generated by CRISPR/Cas9 mediated genome editing.

Research

JoVE Journal

Genetics

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Criação e Double-stranded mediada por RNA silenciamento de genes na Esconder Beetle,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Genética

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

Usando uma técnica de PCR fluorescente-capilar Electroforese em Gel de genótipo CRISPR / cas9 mediada KO mutantes em um formato de elevado rendimento

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

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

Uma metodologia padrão para examinar a mutagenicidade no local como uma função de ponto de mutação reparação catalisada por CRISPR/Cas9 e SsODN em células humanas

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

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Geração de um Gene-interrompeu Streptococcus mutans estirpe sem Gene clonagem

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

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

Integração de alvo CRISPR/Cas9-mediada na Vivo usando uma estratégia de baseada em juntar-se mediada por homologia final

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

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Um protocolo para a produção de Lentivirus vetores Integrase deficiente CRISPR/Cas9-mediada Gene nocaute em dividir células

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

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

Produção eficiente e identificação de CRISPR/Cas9 gerado pelo Gene nocautes no sistema modelo Danio rerio

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

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

Adaptação da hibridação capturar de proteínas da cromatina associada para proteômica para células de mamíferos

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Formaldeído-assistida isolamento de elementos reguladores para acessibilidade de cromatina medida em células de mamíferos

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

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

Disrupção do Gene altamente eficiente de células progenitoras hematopoiéticas murino e humano por 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. ...