Name: a protocol for the production of integrase-deficient lentiviral vectors for crispr/cas9-mediated gene knockout in dividing cells
Uploaded: 2017-12-12T14:00:00
Description: Watch this Scientific Journal Video about A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells at JoVE.com

Vi vil gerne takke afdeling neurobiologi, Duke University School of Medicine og Dekansekretariatet for grundlæggende videnskab, Duke University. Vi takker også medlemmerne af hertug Viral vektor kerne for kommentarer på manuskriptet. Plasmidet pLenti CRISPRv2 var gave fra Feng Zhang (Broad Institute). LV-emballage system herunder plasmider psPAX2, var VSV-G, pMD2.G og pRSV-Rev en slags gave fra Didier Trono (EPFL, Schweiz). Finansiel støtte til dette arbejde blev leveret af universitetet i South Carolina School Of Medicine, give RDF18080-E202 (B.K).

1. dyrkning HEK-293T celler og såning celler for Transfektion
Bemærk: Menneskelige embryonale nyre 293T (HEK-293T) celler dyrkes i DMEM, høje glukose media suppleret med 10% kvæg kalveserum suppleret med jern og vækst initiativtagere og 1 x antibiotikum-antimykotikum løsning (100 x opløsning indeholder 10.000 enheder penicillin 10 mg streptomycin og 25 µg amphotericin B pr. mL). Medierne er også suppleret med 1 x natrium pyruvat, 1 x ikke-essentiel aminosyre mix og 2 mM L-glutamin (stoc...

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	<li>Horvath, P., Barrangou, R. <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,+the+immune+system+of+bacteria+and+archaea.">CRISPR/Cas, the immune system of bacteria and archaea.</a> Science. 327 (5962), 167-170 (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:+RNA-directed+adaptive+immunity+in+bacteria+and+archaea.">CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea.</a> Nat Rev Genet. 11 (3), 181-190 (2010).</li><li>Kim, H., 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=A+guide+to+genome+engineering+with+programmable+nucleases.">A guide to genome engineering with programmable nucleases.</a> Nat Rev Genet. 15 (5), 321-334 (2014).</li><li>Bellec, 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=CFTR+inactivation+by+lentiviral+vector-mediated+RNA+interference+and+CRISPR-Cas9+genome+editing+in+human+airway+epithelial+cells.">CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.</a> Curr Gene Ther. 15 (5), 447-459 (2015).</li><li>Kennedy, E. 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=Suppression+of+hepatitis+B+virus+DNA+accumulation+in+chronically+infected+cells+using+a+bacterial+CRISPR/Cas+RNA-guided+DNA+endonuclease.">Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease.</a> Virology. 476, 196-205 (2015).</li><li>Roehm, P. 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=Inhibition+of+HSV-1+Replication+by+Gene+Editing+Strategy.">Inhibition of HSV-1 Replication by Gene Editing Strategy.</a> Sci Rep. 6, 23146 (2016).</li><li>Wang, 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=CCR5+gene+disruption+via+lentiviral+vectors+expressing+Cas9+and+single+guided+RNA+renders+cells+resistant+to+HIV-1+infection.">CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.</a> PLoS One. 9 (12), e115987 (2014).</li><li>Kantor, B., Bailey, R. M., Wimberly, K., Kalburgi, S. N., Gray, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+gene+transfer+to+the+central+nervous+system.">Methods for gene transfer to the central nervous system.</a> Adv Genet. 87, 125-197 (2014).</li><li>Lebbink, 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=A+combinational+CRISPR/Cas9+gene-editing+approach+can+halt+HIV+replication+and+prevent+viral+escape.">A combinational CRISPR/Cas9 gene-editing approach can halt HIV replication and prevent viral escape.</a> Sci Rep. 7, 41968 (2017).</li><li>Xu, 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=CRISPR/Cas9-Mediated+CCR5+Ablation+in+Human+Hematopoietic+Stem/Progenitor+Cells+Confers+HIV-1+Resistance+In+Vivo.">CRISPR/Cas9-Mediated CCR5 Ablation in Human Hematopoietic Stem/Progenitor Cells Confers HIV-1 Resistance In Vivo.</a> Mol Ther. , (2017).</li><li>Yiu, G., Tieu, E., Nguyen, A. T., Wong, B., Smit-McBride, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Disruption+of+VEGF-A+Expression+in+Human+Retinal+Pigment+Epithelial+Cells+Using+CRISPR-Cas9+Endonuclease.">Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.</a> Invest Ophthalmol Vis Sci. 57 (13), 5490-5497 (2016).</li><li>Kabadi, A. M., Ousterout, D. G., Hilton, I. B., Gersbach, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+CRISPR/Cas9-based+genome+engineering+from+a+single+lentiviral+vector.">Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.</a> Nucleic Acids Res. 42 (19), e147 (2014).</li><li>Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+CRISPR-Cas+nuclease+specificity+using+truncated+guide+RNAs.">Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.</a> Nat Biotechnol. 32 (3), 279-284 (2014).</li><li>Hsu, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases.">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nat Biotechnol. 31 (9), 827-832 (2013).</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><li>Pattanayak, V., 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-throughput+profiling+of+off-target+DNA+cleavage+reveals+RNA-programmed+Cas9+nuclease+specificity.">High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.</a> Nat Biotechnol. 31 (9), 839-843 (2013).</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 (6166), 84-87 (2014).</li><li>Schaefer, K. 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=Unexpected+mutations+after+CRISPR-Cas9+editing+in+vivo.">Unexpected mutations after CRISPR-Cas9 editing in vivo.</a> Nat Methods. 14 (6), 547-548 (2017).</li><li>Choi, J. 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=Lentivirus+pre-packed+with+Cas9+protein+for+safer+gene+editing.">Lentivirus pre-packed with Cas9 protein for safer gene editing.</a> Gene Ther. 23 (7), 627-633 (2016).</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 (2), 440-455 (2014).</li><li>Truong, D. 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=Development+of+an+intein-mediated+split-Cas9+system+for+gene+therapy.">Development of an intein-mediated split-Cas9 system for gene therapy.</a> Nucleic Acids Res. 43 (13), 6450-6458 (2015).</li><li>Nelson, C. E., Gersbach, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Delivery+Vehicles+for+Genome+Editing.">Engineering Delivery Vehicles for Genome Editing.</a> Annu Rev Chem Biomol Eng. 7, 637-662 (2016).</li><li>Jaalouk, D. E., Crosato, M., Brodt, P., Galipeau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+histone+deacetylation+in+293GPG+packaging+cell+line+improves+the+production+of+self-inactivating+MLV-derived+retroviral+vectors.">Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors.</a> Virol J. 3, 27 (2006).</li><li>Ellis, B. L., Potts, P. R., Porteus, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+higher+titer+lentivirus+with+caffeine.">Creating higher titer lentivirus with caffeine.</a> Hum Gene Ther. 22 (1), 93-100 (2011).</li><li>Hoban, M. 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=Delivery+of+Genome+Editing+Reagents+to+Hematopoietic+Stem/Progenitor+Cells.">Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells.</a> Curr Protoc Stem Cell Biol. 36, 1-10 (2016).</li><li>Bayer, 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+large+U3+deletion+causes+increased+in+vivo+expression+from+a+nonintegrating+lentiviral+vector.">A large U3 deletion causes increased in vivo expression from a nonintegrating lentiviral vector.</a> Mol Ther. 16 (12), 1968-1976 (2008).</li><li>Kantor, B., Ma, H., Webster-Cyriaque, J., Monahan, P. E., Kafri, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+activation+of+unintegrated+HIV-1+genomes+by+gut-associated+short+chain+fatty+acids+and+its+implications+for+HIV+infection.">Epigenetic activation of unintegrated HIV-1 genomes by gut-associated short chain fatty acids and its implications for HIV infection.</a> Proc Natl Acad Sci U S A. 106 (44), 18786-18791 (2009).</li><li>Ortinski, P. I., O'Donovan, B., Dong, X., Kantor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrase-Deficient+Lentiviral+Vector+as+an+All-in-One+Platform+for+Highly+Efficient+CRISPR/Cas9-Mediated+Gene+Editing.">Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing.</a> Mol Ther Methods Clin Dev. 5, 153-164 (2017).</li><li>Kantor, 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=Notable+reduction+in+illegitimate+integration+mediated+by+a+PPT-deleted,+nonintegrating+lentiviral+vector.">Notable reduction in illegitimate integration mediated by a PPT-deleted, nonintegrating lentiviral vector.</a> Mol Ther. 19 (3), 547-556 (2011).</li><li>Berkhout, B., Verhoef, K., van Wamel, J. L., Back, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+instability+of+live,+attenuated+human+immunodeficiency+virus+type+1+vaccine+strains.">Genetic instability of live, attenuated human immunodeficiency virus type 1 vaccine strains.</a> J Virol. 73 (2), 1138-1145 (1999).</li><li>Gomez-Gonzalo, 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=The+hepatitis+B+virus+X+protein+induces+HIV-1+replication+and+transcription+in+synergy+with+T-cell+activation+signals:+functional+roles+of+NF-kappaB/NF-AT+and+SP1-binding+sites+in+the+HIV-1+long+terminal+repeat+promoter.">The hepatitis B virus X protein induces HIV-1 replication and transcription in synergy with T-cell activation signals: functional roles of NF-kappaB/NF-AT and SP1-binding sites in the HIV-1 long terminal repeat promoter.</a> J Biol Chem. 276 (38), 35435-35443 (2001).</li><li>Kim, Y. 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=Artificial+zinc+finger+fusions+targeting+Sp1-binding+sites+and+the+trans-activator-responsive+element+potently+repress+transcription+and+replication+of+HIV-1.">Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.</a> J Biol Chem. 280 (22), 21545-21552 (2005).</li><li>Ortinski, P. I., Lu, C., Takagaki, K., Fu, Z., Vicini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+distinct+alpha+subunits+of+GABAA+receptor+regulates+inhibitory+synaptic+strength.">Expression of distinct alpha subunits of GABAA receptor regulates inhibitory synaptic strength.</a> J Neurophysiol. 92 (3), 1718-1727 (2004).</li><li>Van Lint, 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=Transcription+factor+binding+sites+downstream+of+the+human+immunodeficiency+virus+type+1+transcription+start+site+are+important+for+virus+infectivity.">Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity.</a> J Virol. 71 (8), 6113-6127 (1997).</li><li>Van Lint, C., Ghysdael, J., Paras, P., Burny, A., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptional+regulatory+element+is+associated+with+a+nuclease-hypersensitive+site+in+the+pol+gene+of+human+immunodeficiency+virus+type+1.">A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1.</a> J Virol. 68 (4), 2632-2648 (1994).</li><li>Xu, W., Russ, J. L., Eiden, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+residual+promoter+activity+in+gamma-retroviral+self-inactivating+(SIN)+vectors.">Evaluation of residual promoter activity in gamma-retroviral self-inactivating (SIN) vectors.</a> Mol Ther. 20 (1), 84-90 (2012).</li><li>. Testing for Replication Competent Retrovirus (RCR)/Lentivirus (RCL) in Retroviral and Lentiviral Vector Based Gene Therapy Products - Revisiting Current FDA Recommendations Available from: <a target="_blank" href="https://sites.duke.edu/dvvc/files/2016/05/FDA-recommendation-for-RCR-testing.pdf">https://sites.duke.edu/dvvc/files/2016/05/FDA-recommendation-for-RCR-testing.pdf</a> (2017)</li><li>. Biosafety in Microbiological and Biomedical Laboratories Available from: <a target="_blank" href="https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf">https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf</a> (2017)</li><li>Dull, 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=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J Virol. 72 (11), 8463-8471 (1998).</li><li>Liu, X. 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=Editing+DNA+Methylation+in+the+Mammalian+Genome.">Editing DNA Methylation in the Mammalian Genome.</a> Cell. 167 (1), 233-247 (2016).</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=In+vivo+genome+editing+using+Staphylococcus+aureus+Cas9.">In vivo genome editing using Staphylococcus aureus Cas9.</a> Nature. 520 (7546), 186-191 (2015).</li><li>Zetsche, 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=Cpf1+is+a+single+RNA-guided+endonuclease+of+a+class+2+CRISPR-Cas+system.">Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.</a> Cell. 163 (3), 759-771 (2015).</li></ol>

IDLVs begyndt at dukke op som køretøjet af valg til i vivo gen-redigering, især i forbindelse med genetiske sygdomme, i vid udstrækning på grund af lav risiko for mutagenese forbundet med disse vektorer i forhold til at integrere levering platforme22 , 28. i det aktuelle manuskript, søgte vi at detalje den protokol, der er forbundet med produktionen af den forbedrede All-in-one IDLV-CRISPR/Cas9 system, der var for nylig udviklet i vores laboratoriu...

Præcise gen redigering udgør hjørnestenen i store biomedicinsk forskud, der vedrører udviklingen af nye strategier til at tackle genetiske sygdomme. På forkant med gen-redigering teknologier er den metode, der er afhængige af brugen af clustered regularly -jegnterspaced short palindromic repeats (CRISPR) / Cas9 system, der var i første omgang identificeret som en komponent af bakteriel immunitet mod invasionen af viral genetiske materiale (gennemgik i referencer1,2). En stor fordel af ordningen med CRISPR/Cas9 over andre gen-redaktion værktøj, såsom zink-finger nukleaser (ZFNs) og transkription aktivere-lignende effektor nukleaser (TALENs) (gennemgik i reference3), er den relative enkelhed af plasmid design og opførelse af CRISPR komponenter — en funktion, der har drevet udvidelse af gen-redigering fra nogle få specialiserede laboratorier til en meget bredere forskning samfund. Derudover har enkelhed af CRISPR/Cas9 programmering og dets evne til at multiplex mål anerkendelse yderligere næret sin popularitet som en omkostningseffektiv og nem at bruge teknologi. Blandt de forskellige metoder til rådighed for forskerne til at levere sådanne gen-redigering komponenter til celler, stadig virale vektorer langt den mest populære og effektive system.
Lentiviral vektorer (LVs) er dukket op som køretøjet af valg til at levere komponenter i CRISPR/Cas9 system i vivo for forskellige applikationer4,5,6,7. Flere nøglen egenskaber gør LVs et populært valg for denne proces, herunder deres evne til at inficere både delende og ikke-dividere celler, lav immunogenicitet og minimal cellulære toksicitet (gennemgik i reference8). Som et resultat, har LV-medieret genterapi været ansat i behandlinger af smitsomme sygdomme, såsom HIV-1, HBV og HSV-1, og korrektion af fejl underliggende menneskelige arvelige sygdomme som cystisk fibrose og neo-kar makuladegeneration 4 , 5 , 7 , 9 , 10 , 11. Desuden, LVs har ændret effektivt for at udføre multiplex gen redigering på særskilte genomisk loci ved hjælp af en enkelt vektor system12.
Men den iboende egenskab af LVs at integrere i host-genom kan være mutagene og ofte handicap for deres nytte som transgen levering køretøjer, især i klinisk indstillinger. Desuden, da stabilt integreret LVs udtrykke deres transgener på bæredygtigt høje niveauer, dette system er dårligt egnet til levering af gen-redigering komponenter som CRISPR/Cas9; overekspression af Cas9-guide RNA (gRNA) og lignende proteiner som ZFNs, er forbundet med forhøjede niveauer af off-target effekter, som omfatter uønskede mutationer13,14,15,16 , 17 og kan potentielt forbedre cytotoksicitet18. For at opnå præcise gen-redigering med minimal off target effekter, er det derfor afgørende, at design-systemer, der giver mulighed for den forbigående udtryk af genet redigering komponenter.
I de seneste år, er blevet udviklet en række forskellige leveringsplatforme forbigående udtrykke CRISPR/Cas9 i celler16,19,20,21 (gennemgik i reference22). Disse omfatter metoder, der er baseret på direkte at indføre renset Cas9 sammen med passende guide RNA'er i celler, som viste sig at være mere effektive på målrettede gen-redigering i forhold til plasmid-medieret Transfektion16. Undersøgelser har vist, at ribonucleoprotein (RNP) komplekser bestående af guide RNA/Cas9 partikler er hurtigt vendt efter mægle DNA kavalergang på deres mål, tyder på, at kortsigtede udtryk for disse komponenter er tilstrækkelige til at opnå robust gen redigering16. Tænkes, ikke integrere viral vektor platforme såsom adeno-associeret virale vektorer (AAVs) kan give et rentabelt alternativ for at levere gen-redigering maskiner til cellerne. Desværre, AAV capsids besidder betydeligt lavere emballage kapacitet end LVs (< 5kb), der alvorligt hæmmer deres evne til at pakke den multi-komponent CRISPR toolkit inden for en enkelt vektor (gennemgik i reference8). Det er værd at bemærke, at tilsætning af stoffer, der hæmmer Histon deacetyltransferase (f.eks., natrium butyrat23) eller hindre cellecyklus (fx, koffein24) har vist sig at øge lentiviral titers. Trods de seneste fremskridt, er forbigående udtryk systemer udviklet hidtil stadig hæmmet af flere mangler, såsom lavere produktionseffektivitet, hvilket fører til nedsat viral titers og lav transduktion effektivitet af vira genereret gennem sådanne tilgange25.
Integrase-mangelfuld lentiviral vektorer (IDLVs) repræsenterer et stort fremskridt i udviklingen af gen-levering køretøjer, som de kombinerer den emballage kapacitet af LVs med den ekstra fordel af AAV-lignende episomal vedligeholdelse i celler. Disse funktioner hjælpe IDLVs i vid udstrækning omgå de store spørgsmål i forbindelse med integration af vektorer, vis-à-vis kontinuerlig overekspression af potentielt genotoksiske elementer og integration-medieret mutagenicitet. Tidligere blev det påvist, at IDLVs kan ændres med held til at forbedre episomal gen expression26,27. Med hensyn til IDLV-medieret CRISPR/Cas9 levering begrænser lav produktion titers og lavere udtryk af episome-bårne genomer i forhold til integrase-dygtige lentiviral systemer deres nytte som Bonafide værktøjer til leverer genom-redigering transgene konstruktioner. Vi har for nylig vist, at både transgen udtryk og viral titers tilknyttet IDLV produktion er betydeligt forbedret ved inddragelse af bindende websteder for transkriptionsfaktor Sp1 inden for viral udtryk kassette28. De modificerede IDLVs understøttes håndfast CRISPR-medieret gen redigering både in vitro- (i HEK-293T celler) og i vivo (i post mitotiske hjernen neuroner), mens inducerende minimal off target mutationer i forhold til de tilsvarende ICLV-medieret systemer28. Samlet set udviklede vi en roman, kompakt, All-in-one CRISPR toolkit transporteres på en IDLV platform og skitseret de forskellige fordele ved at bruge sådanne fremføringsmiddel for forbedret gen redigering.
Her, er produktion protokol af IDLV-CRISPR/Cas9-systemet beskrevet, herunder de forskellige trin involveret i montage, rensning, koncentration og titrering af IDLVs, samt strategier til at validere gen-redigering effekten af disse vektorer. Denne protokol er let skalerbar til at opfylde behovene hos forskellige efterforskere og er designet til at kunne generere LV vektorer med titers i rækken af 1 x 1010 transducing enheder (TU) / mL. Vektorer genereres via denne protokol kan udnyttes til at effektivt inficere flere forskellige celletyper, herunder vanskeligt at transduce embryonale stamceller, hæmatopoietisk celler (T-celler og makrofager) og kulturperler og in vivo- injiceres neuroner. Derudover er protokollen lige så velegnet til produktion af integrase-kompetente lentiviral vektorer i lignende mængder.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56915/56915fig1.jpg"> Figur 1: IDLV emballage. (a) skematisk af vildtype integrase protein (b) den modificerede plasmid stammer fra psPAX2 (Se metoder, plasmid konstruktion for detaljer). Repræsentant Agarosen gel billede af kloner screenet for muterede integrase kloner. DNA-prøver udarbejdet ved hjælp af en standard plasmid DNA isoleret mini kit blev analyseret af fordøjelse med EcoRV og SphI. Korrekt fordøjet klon (nummer 5, stiplet rød boks) blev yderligere bekræftet ved direkte (Sanger) sekventering substitutionsrelevante D64E i INT. Integrase-mangelfuld emballage kassette blev opkaldt pBK43. (c) skematisk af forbigående Transfektion protokol ansat til at generere IDLV-CRISPR/Cas9 vektorer, viser 293T celler transfekteret med VSV-G, emballage og transgen kassetter (Sp1-CRISPR/Cas9 All-in-one plasmid). Viruspartikler, der bud ud fra cellens membran indeholder den fuld længde RNA af vektoren (udtrykt fra transgene kassette). Den anden generation af IDLV-emballagesystem blev brugt, som omfatter de lovgivningsmæssige proteiner Tat og rev Rev udtryk er yderligere suppleres fra en separat kassette (RSV-REV-plasmid). Kongerige: LTR-lang-terminal gentage, VSV-G, vesikulær stomatitis virus G-protein, pCMV-cytomegalovirus promotor; Rous sarkom virus (RSV) promotor; RRE-(Rev svar Element). Andre regulerende elementer på udtryk kassette omfatter Sp1-bindingssteder, Rev Response element (RRE), Woodchuck Hepatitis Virus posttranskriptionelle regulerende Element (WPRE), en kerne-brudforlængelse faktor 1α promotor (EFS-NC), vektor emballage element ψ (psi), menneskelige Cytomegalovirus (hCMV) promotor og menneskelige U6 promotor. <a href="//cloudflare.jove.com/files/ftp_upload/56915/56915fig1large.jpg" target="_blank">Venligst klik her for at se en større version af dette tal.</a>

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XPN-80 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A99839</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra 25R tabletop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>369434</td>
			<td></td>
		</tr>
		<tr>
			<td>xMark Microplate Absorbance plate reader</td>
			<td>Bio-Rad</td>
			<td>1681150</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS</td>
			<td>Becton Dickinson</td>
			<td>338960</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Leica</td>
			<td>DM IRB2</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45-&#956;m filter unit, 500mL</td>
			<td>Corning</td>
			<td>430773</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical bottom ultracentrifugation tubes</td>
			<td>Seton Scientific</td>
			<td>5067</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube adapters</td>
			<td>Seton Scientific</td>
			<td>PN 4230</td>
			<td></td>
		</tr>
		<tr>
			<td>SW32Ti swinging-bucket rotor</td>
			<td>Beckman Coulter</td>
			<td>369650</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430291</td>
			<td></td>
		</tr>
		<tr>
			<td>High-binding 96-well plates</td>
			<td>Corning</td>
			<td>3366</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm TC-Treated Cell Culture dishes with 20 mm Grid</td>
			<td>Corning</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;M filter unit, 1L</td>
			<td>Corning</td>
			<td>430513</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (50)</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture pipettes, 5 mL</td>
			<td>Corning</td>
			<td>4487</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture pipettes, 10 mL</td>
			<td>Corning</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture pipettes, 25 mL</td>
			<td>Corning</td>
			<td>4489</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer with cover slips</td>
			<td>Cole-Parmer</td>
			<td>UX-79001-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell culture reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human embryonic kidney 293T (HEK 293T) cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>293FT cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose media</td>
			<td>Gibco</td>
			<td>11965</td>
			<td></td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH30087.04</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic solution, 100X</td>
			<td>Sigma Aldrich</td>
			<td>A5955-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>S8636-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acid (NEAA)</td>
			<td>Hyclone</td>
			<td>SH30087.04</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>BES (N, N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid)</td>
			<td>Sigma Aldrich</td>
			<td>B9879 - BES</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G1800-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>p24 ELISA reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal anti-p24 antibody</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>3537</td>
			<td></td>
		</tr>
		<tr>
			<td>HIV-1 standards</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>SP968F</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-p24 antibody</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>SP451T</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit horseradish peroxidase IgG</td>
			<td>Sigma Aldrich</td>
			<td>12-348</td>
			<td>Working concentration 1:1500</td>
		</tr>
		<tr>
			<td>Normal mouse serum, Sterile, 500mL</td>
			<td>Equitech-Bio</td>
			<td>SM30-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum, Sterile, 10mL</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr>
			<td>TMB peroxidase substrate</td>
			<td>KPL</td>
			<td>5120-0076</td>
			<td>Working concentration 1:10,000</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>lentiCRISPR v2</td>
			<td>Addgene</td>
			<td>52961</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Restriction enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BsrGI</td>
			<td>New England Biolabs</td>
			<td>R0575S</td>
			<td></td>
		</tr>
		<tr>
			<td>BsmBI</td>
			<td>New England Biolabs</td>
			<td>R0580S</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRV</td>
			<td>New England Biolabs</td>
			<td>R0195S</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI</td>
			<td>New England Biolabs</td>
			<td>R0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>PacI</td>
			<td>New England Biolabs</td>
			<td>R0547S</td>
			<td></td>
		</tr>
		<tr>
			<td>SphI</td>
			<td>New England Biolabs</td>
			<td>R0182S</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for the production of integrase-deficient lentiviral vectors for crispr/cas9-mediated gene knockout in dividing cells

Lentiviral vektorerne er et ideelt valg til at levere gen-redigering komponenter til celler på grund af deres evne til stabilt transducing en bred vifte af celler og mægle høje niveauer af genekspression. Men deres evne til at integrere i vært celle genom øger risikoen for pattedyrsceller mutagenicitet og dermed giver anledning til sikkerhedsmæssige betænkeligheder og begrænser deres anvendelse i kliniske indstillinger. Yderligere, den vedvarende udtryk for gen-redigering komponenter leveret af disse integration-kompetente lentiviral vektorer (ICLVs) øger sandsynligheden for promiskuøs gen målretning. Som et alternativ, er blevet udviklet en ny generation af integrase-mangelfuld lentiviral vektorer (IDLVs), der omhandler mange af disse bekymringer. Her produktion protokollen af en ny og forbedret IDLV platform for CRISPR-medieret gen redigering og listen trin involveret i rensning og koncentrationen af disse vektorer er beskrevet og deres transduktion og gen-redigering effektivitet ved hjælp af HEK-293T celler blev demonstreret. Denne protokol er let skalerbar og kan bruges til at generere høj titer IDLVs, der er i stand til at transducing celler in vitro og in vivo. Desuden, denne protokol kan nemt tilpasses til produktion af ICLVs.

Validering af IDLV-CRISPR/Cas9 vektorer knockout-effektivitet Vi brugte normal god landbrugspraksis-udtrykker 293T celler som en model til at validere effektiviteten af CRISPR/Cas9-medieret gen knockout. Normal god landbrugspraksis + celler blev genereret af transduktion af HEK-293T celler med pLenti-NGL (vBK201a) på et MOI af 0,5 (figur 3b, "no-virus" panel). SgRNA-til-normal god landbrugspraksis/Cas9 All-in-one vektor kassette var pakke...

Watch this Scientific Journal Video about A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells at JoVE.com

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

En protokol til produktion af Integrase-mangelfuld Lentiviral vektorer for CRISPR/Cas9-medieret gen Knockout i dividere celler

Vi beskrive produktion strategien integrase-mangelfuld lentiviral vektorer (IDLVs) som køretøjer til at levere CRISPR/Cas9 til cellerne. Med en evne til at mægle, hurtig og robust gen redigering i celler, IDLVs præsentere et sikrere og lige så effektive vektor platform for gen levering i forhold til integrase-kompetente vektorer.

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 ...

Research

JoVE Journal

Genetics

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

Under anvendelse af et fluorescerende PCR-kapillær gelelektroforese Teknik til genotype CRISPR / Cas9-medieret Knockout Mutanter i et format med høj kapacitet

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

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 ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

En universel protokol for storstilet gRNA bibliotek produktion fra enhver kilde, DNA

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

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 ...

Production and Purification of Baculovirus for Gene Therapy Application

Produktion og oprensning af Baculovirus for genterapi ansøgning

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

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. ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

Lentiviral medieret produktion af Transgene mus: en enkel og meget effektiv metode til direkte undersøgelse af grundlæggerne

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

Høj overførselshastighed identifikation af regulerende gensekvenser ved hjælp af næste generations sekventering af cirkulære kromosom kropsbygning Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

In vivo anvendelse af sen-kontrol-System for Manipulation af endogene genekspression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...