We thank previous members of our laboratory and scientist Serif Senturk for previous work. We thank Danilo Segovia for critically reading this manuscript. This study was possible and supported by Swim Across America and the National Cancer Institute Cancer Target Discovery and Development Center program.

1.The DD-Cas9 vector
<ol>
	<li>Obtain DD-Cas9 vector from Addgene (DD-Cas9 with filler sequence and Venus (EDCPV), Plasmid 90085). 
	&#8203;NOTE: This is a lentiviral DD-Cas9 plasmid with a U6 promoter that drives the single guide RNA (sgRNA) transcription while the EFS promoter drives the DD-Cas9 transcription. The DYKDDDDK sequence (flag-tag) is present at the C-terminal of Cas9 followed by 2A self-cleaving peptide (P2A) that separates DD-Cas9 and modified fluorescent protein Venus (mVenus).</li>
</ol>
<p class=...

<ol>
	<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, 816-821 (2012).</li><li>Al-Attar, S., Westra, E. R., van der Oost, J., Brouns, 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=Clustered+regularly+interspaced+short+palindromic+repeats+(CRISPRs):+the+hallmark+of+an+ingenious+antiviral+defense+mechanism+in+prokaryotes.">Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes.</a> Biological Chemistry. 392, 277-289 (2011).</li><li>Hsu, P. D., Lander, E. S., 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=Development+and+applications+of+CRISPR-Cas9+for+genome+engineering.">Development and applications of CRISPR-Cas9 for genome engineering.</a> Cell. 157, 1262-1278 (2014).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</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=Advances+in+CRISPR-Cas9+genome+engineering:+lessons+learned+from+RNA+interference.">Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference.</a> Nucleic Acids Research. 43, 3407-3419 (2015).</li><li>Zhang, J., Chen, L., Zhang, J., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+Inducible+CRISPR/Cas+Systems.">Drug Inducible CRISPR/Cas Systems.</a> Computational and Structural Biotechnology Journal. , (2019).</li><li>Wade, M. <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+Silencing+Using+the+CRISPR-Cas9+System:+A+Review+of+the+Benefits+and+Challenges.">High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges.</a> Journal of Biomolecular Screening. 20, 1027-1039 (2015).</li><li>Egeler, E. L., Urner, L. M., Rakhit, R., Liu, C. W., Wandless, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-switchable+substrates+for+a+ubiquitin-proteasome+system.">Ligand-switchable substrates for a ubiquitin-proteasome system.</a> Journal of Biological Chemistry. 286, 31328-31336 (2011).</li><li>Cao, 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=An+easy+and+efficient+inducible+CRISPR/Cas9+platform+with+improved+specificity+for+multiple+gene+targeting.">An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting.</a> Nucleic Acids Research. 44, 149 (2016).</li><li>Oakes, B. 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=Profiling+of+engineering+hotspots+identifies+an+allosteric+CRISPR-Cas9+switch.">Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.</a> Nature Biotechnology. 34, 646-651 (2016).</li><li>Kamiyama, 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=Versatile+protein+tagging+in+cells+with+split+fluorescent+protein.">Versatile protein tagging in cells with split fluorescent protein.</a> Nature Communications. 7, 11046 (2016).</li><li>Roper, 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=In+vivo+genome+editing+and+organoid+transplantation+models+of+colorectal+cancer+and+metastasis.">In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.</a> Nature Biotechnology. 35, 569 (2017).</li><li>Chu, B. 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=Recent+progress+with+FKBP-derived+destabilizing+domains.">Recent progress with FKBP-derived destabilizing domains.</a> Bioorganic &amp; Medicinal Chemistry Letters. 18 (22), 5941-5944 (2008).</li><li>Banaszynski, L. A., Sellmyer, M. A., Contag, C. H., Wandless, T. J., Thorne, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+control+of+protein+stability+and+function+in+living+mice.">Chemical control of protein stability and function in living mice.</a> Nature Medicine. 14, 1123-1127 (2008).</li><li>Banaszynski, L. A., Chen, L. C., Maynard-Smith, L. A., Ooi, A. G., Wandless, T. 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+rapid,+reversible,+and+tunable+method+to+regulate+protein+function+in+living+cells+using+synthetic+small+molecules.">A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules.</a> Cell. 126, 995-1004 (2006).</li><li>Sentmanat, M. F., Peters, S. T., Florian, C. P., Connelly, J. P., Pruett-Miller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Survey+of+Validation+Strategies+for+CRISPR-Cas9+Editing.">A Survey of Validation Strategies for CRISPR-Cas9 Editing.</a> Scientific Reports. 8, 888 (2018).</li><li>Hua, Y., Wang, C., Huang, J., Wang, K. <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+and+efficient+method+for+CRISPR/Cas9-induced+mutant+screening.">A simple and efficient method for CRISPR/Cas9-induced mutant screening.</a> Journal of Genetics and Genomics. 44 (4), 207-213 (2017).</li><li>Guschin, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+general+assay+for+monitoring+endogenous+gene+modification.">A rapid and general assay for monitoring endogenous gene modification.</a> Methods in Molecular Biology. 649, 247-256 (2010).</li><li>Senturk, 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=Rapid+and+tunable+method+to+temporally+control+gene+editing+based+on+conditional+Cas9+stabilization.">Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.</a> Nature Communications. 8, 14370 (2017).</li><li>McJunkin, 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=Reversible+suppression+of+an+essential+gene+in+adult+mice+using+transgenic+RNA+interference.">Reversible suppression of an essential gene in adult mice using transgenic RNA interference.</a> Proceedings of the National Academy of Sciences. 108, 7113 (2011).</li><li>Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule-triggered+Cas9+protein+with+improved+genome-editing+specificity.">Small molecule-triggered Cas9 protein with improved genome-editing specificity.</a> Nature Chemical Biology. 11, 316-318 (2015).</li><li>Dow, 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=Inducible+in+vivo+genome+editing+with+CRISPR-Cas9.">Inducible in vivo genome editing with CRISPR-Cas9.</a> Nature Biotechnology. 33, 390-394 (2015).</li><li>Wright, A. 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=Rational+design+of+a+split-Cas9+enzyme+complex.">Rational design of a split-Cas9 enzyme complex.</a> Proceedings of the National Academy of Sciences of the United States of America. 112, 2984-2989 (2015).</li><li>Albrecht, 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=Comparison+of+Lentiviral+Packaging+Mixes+and+Producer+Cell+Lines+for+RNAi+Applications.">Comparison of Lentiviral Packaging Mixes and Producer Cell Lines for RNAi Applications.</a> Molecular Biotechnology. 57, 499-505 (2015).</li><li>Froschauer, A., Kube, L., Kegler, A., Rieger, C., Gutzeit, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+Protein+Stabilization+In+Vivo+Mediated+by+Shield-1+in+Transgenic+Medaka.">Tunable Protein Stabilization In Vivo Mediated by Shield-1 in Transgenic Medaka.</a> PLOS One. , (2015).</li><li>Sellmyer, M. 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=Intracellular+context+affects+levels+of+a+chemically+dependent+destabilizing+domain.">Intracellular context affects levels of a chemically dependent destabilizing domain.</a> PLOS One. 7 (9), 43297 (2012).</li><li>Iwamoto, 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+general+chemical+method+to+regulate+protein+stability+in+the+mammalian+central+nervous+system.">A general chemical method to regulate protein stability in the mammalian central nervous system.</a> Chemistry &amp; Biology. 17 (9), 981-988 (2010).</li><li>Tai, 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=Destabilizing+domains+mediate+reversible+transgene+expression+in+the+brain.">Destabilizing domains mediate reversible transgene expression in the brain.</a> PLOS One. 7 (9), 46269 (2012).</li><li>Auffenberg, 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=Remote+and+reversible+inhibition+of+neurons+and+circuits+by+small+molecule+induced+potassium+channel+stabilization.">Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization.</a> Scientific Reports. 6, 19293 (2016).</li></ol>

The CRISPR/Cas9 technology has revolutionized the capability of functionally interrogate genomes2. However, the inactivation of genes often results in cell lethality, functional deficits, and developmental defects, limiting the utility of such approaches for studying gene functions7. Additionally, constitutive expression of Cas9 may result in toxicity and the generation of off-target effects6. Different approaches have been developed to temporally co...

CRISPR-Cas9 which stands for &#34;clustered regularly interspaced short palindromic repeats-associated protein 9&#34; was first discovered as part of studies on bacterial adaptive immunity1,2. Today, CRISPR/Cas9 has become the most recognized tool for programmable gene editing and different iterations of the system have been developed to allow transcriptional and epigenetic modulations3. This technology enables the highly precise genetic manipulation of almost any sequence of DNA4.
The essential components of any CRISPR gene editing are a customizable guide RNA sequence and the Cas9 nuclease5. The RNA guide binds to the target-complementary sequence in the DNA, directing the Cas9 nuclease to perform a double-strand break at a specific point in the genome3,4. The resulting cleavage site is then repaired by non-homologous end-joining (NHEJ) or homology-directed repair (HDR), with the consequent introduction of changes in the targeted DNA sequence5.
CRISPR/Cas9 based gene editing is easy to use, and relatively inexpensive compared to previous gene-editing techniques and it has been proven to be both efficient and robust in a multiplicity of systems2,4,5. Yet, the system presents some limitations. The constitutive expression of Cas9 has often been shown to result in an increased number of off-targets and high cell toxicity4,6,7,8. Additionally, the constitutive targeting of essential and cell survival genes by Cas9 takes away from its ability to perform certain types of functional studies such as kinetic studies of cell death7.
Different inducible or conditionally controlled CRISPR-Cas9 tools have been developed to address those issues6, such as Tet-ON and Tet-Off9; site-specific recombination10; chemically-induced proximity11; intein dependent splicing3; and 4-Hydroxytamoxifen Estrogen Receptor (ER) based nuclear localization systems12. In general, most of these procedures (intein splicing and chemically induced proximity split systems) do not offer reversible control, present a very slow kinetic response to drug treatment (Tet-On/Off system), or are not amenable to high-throughput manipulation6.
To address these limitations we developed a novel toolkit that not only provides fast and robust temporal-controlled gene editing but also ensures traceability, tunability, and amenability to high throughput gene manipulation. This novel technology can be used in cell lines, organoids, and animal models. Our system is based on an engineered domain, when fused to Cas9, it induces its rapid degradation. However, it can be rapidly stabilized with a highly selective, non-toxic, cell-permeable small molecule. More specifically, we engineered the human FKBP12 mutant &#34;destabilizing domain&#34; (DD) to Cas9, marking Cas9 for rapid and constitutive degradation via the ubiquitin-proteasome system when expressed in mammalian cells13. The DD synthetic ligand, Shield-1 can stabilize DD conformation, thereby preventing the degradation of proteins fused to DD (such as Cas9) in a very efficient manner, and with a fast kinetic response14,15. Of note, Shield-1 binds with three orders of magnitude tighter to the mutant FKBP12 than to its wild-type counterpart14.
The DD-Cas9/Shield-1 pair can be used to study the systematic identification and characterization of essential genes in cultured cells and animal models as we previously showed by conditionally targeting the CypD gene, which plays an important role in the metabolism of mitochondria; EGFR, a key player in oncogenic transformation; and Tp53, a central gene in DNA damage response. In addition to temporally and conditionally controlled gene editing, another advantage of the method is that the stabilization&#160;of DD-Cas9 is independent of its transcription. This feature enables co-expression, under the same promoter, of traceable markers as well as recombinases, such as the estrogen receptor-dependent recombinase, CREER. In this work, we show how our method can be successfully used in vitro, to conditionally target for example, DNA replication gene, RPA3.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100mM DTT</td>
			<td>Thermosfisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X FastDigest buffer</td>
			<td>Thermosfisher</td>
			<td>B64</td>
			<td></td>
		</tr>
		<tr>
			<td>10X T4 Ligation Buffer</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>colorimetric BCA kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, glutaMax</td>
			<td>Thermo Fisher</td>
			<td>10566024</td>
			<td></td>
		</tr>
		<tr>
			<td>FastAP</td>
			<td>Thermosfisher</td>
			<td>EF0654</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest BsmBI</td>
			<td>Thermosfisher</td>
			<td>FD0454</td>
			<td></td>
		</tr>
		<tr>
			<td>Flag [M2] mouse mAb</td>
			<td>Sigma</td>
			<td>F1804-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA extraction kit</td>
			<td>Macherey Nagel</td>
			<td>740952.1</td>
			<td></td>
		</tr>
		<tr>
			<td>lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>oligonucleotides</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma Aldrich</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR &#38; Gel Cleanup Kit</td>
			<td>Qiagen</td>
			<td>28506</td>
			<td></td>
		</tr>
		<tr>
			<td>secondary antibodies</td>
			<td>LICOR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shield-1</td>
			<td>Cheminpharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stbl3 competent bacterial cells</td>
			<td>Thermofisher</td>
			<td>C737303</td>
			<td></td>
		</tr>
		<tr>
			<td>SURVEYOR Mutation Detection Kit</td>
			<td>Transgenomic/IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>NEB</td>
			<td>M0273S</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-tubulin [DM1A] mouse mAb</td>
			<td>Millipore</td>
			<td>CP06-100UG</td>
			<td></td>
		</tr>
	</tbody>
</table>

a new toolkit for evaluating gene functions using conditional cas9 stabilization

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.

To enable the conditional expression of Cas9, we developed a dual lentiviral vector construct consisting of a U6-driven promoter to constitutively express sgRNA, and an EF-1&#945; core promoter to drive the expression of the DD-Cas9 fusion protein (Figure 1A)19. As a paradigm to illustrate the robustness and efficiency of the system, we transduced the lung carcinomatous A549 cell line with the lentiviral construct. The levels of Cas9 i...

Watch this Scientific Journal Video about A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization at JoVE.com

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Here, we describe a genome-editing tool based on the temporal and conditional stabilization of clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) under the small molecule, Shield-1. The method can be used for cultured cells and animal models.

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory ...

This method provides fast, temperature controlled CRISPR-Cas9 gene editing under a small molecule, Shield-1. It also offers less off-target effects, lower cell toxicity, and also higher internally controlled gene editing when comparing it to constitutive activation of Cas9. The main advantage of this particle is that it can be used for characterization of essential genes, as well as tumor survival genes.Destabilization of DD-Cas9 is independent of its expression, and this allows the gene of interest to be co-expressed under the same promoter. This method can be easily applied to in vivo and in vitro studies. Shield-1 can also penetrate through blood-brain barrier, which making it easy also to study genes that are involved in brain development.Evenly seed healthy HEK293T cells at a density of one to two million cells per 10 centimeter tissue culture plate by using 10 milliliters of glucose DMEM with 10%filtered FBS. Incubate the cells in the tissue culture incubator at 5%carbon dioxide and 37 degrees Celsius for 20 hours. On the next day, confirm that the cells reached 70%confluency by brightfield microscopy and that they are evenly dispersed across the plate.Change media one hour before the transfection with a final volume of 10 milliliters. Prepare two tubes with 500 microliters of warm media. Add 25 microliters of transfection reagent to tube one, and incubate at room temperature for five minutes.To the other tube, add a mixture of plasmids. Mix tube one with tube two to form a transfection mixture, and incubate at room temperature for 20 minutes. Add the transfection mixture dropwise to the HEK293T cells and incubate the plate in the tissue culture incubator.After 18 hours, carefully change the media to 10 milliliters of fresh DMEM with 10%FBS to remove the transfection reagent. Incubate the cells for the next 48 hours. After 48 hours, collect the supernatant with a 10 milliliter syringe and pass it through a 0.45 micrometer filter.Aliquot the virus supernatant and store it at minus 80 degrees Celsius. For determining virus titer, seed 5 million HEK293T cells per well into two six-well plates. Incubate both plates at 37 degrees Celsius and 5%carbon dioxide overnight to reach 50 to 60%confluency.On the next day, use one of the six-well plates to count the cells in the six wells. Thaw the virus aliquot that will be used to perform the serial dilution and add eight micrograms per milliliter of polybrene reagent, prepare DMEM with 10%FBS for serial dilution of the virus, and add the polybrene reagent. Prepare two milliliters of tenfold serial dilutions of the lentivirus from 0.1 to 0.0001 in polybrene containing media.Remove media from the six-well plate and add one milliliter of viral dilutions to the wells, leaving one well with media alone as a negative control and one well with 100%virus. Incubate the cells for 24 hours. On the next day, remove the media with the virus from the six-well plates and change it to two milliliters of fresh DMEM with 10%FBS.Incubate the cells for 48 to 72 hours, observing GFP with a fluorescent microscope every day. After 48 to 72 hours, detach the cells and resuspend them in max buffer. Use a flow cytometer to determine the percentage of GFP expression and calculate the virus titer using the formula given in the text manuscript.Plate the cell line of interest in a 10 centimeter plate and incubate overnight to reach confluency of 50 to 60%Then infect the cells with 500 to 2, 000 microliters of virus particles in a 10 milliliter total volume of culture medium. Incubate the viral media for 24 hours. On the next day, change the viral media to culture media, and determine the percentage of GFP positive cells by using flow cytometry.Select GFP positive cells by using BD FACSAria II for cell sorting. Expand positively selected cells and freeze the stock. Plate the positively selected cells and untransduced cells in 12-well plates separately, 24 hours after infection.Wait until the cells attach and change the media to cell culture media containing 200 nanomolar Shield-1. Replace the media in the plates with transduced and untransduced cells, leaving two wells per plate with regular media as a negative control. Incubate and extract proteins from each well at zero, two, six, 12, 24, 48, and 72 hours after adding Shield-1 to the wells.Then remove the media with Shield-1 from the rest of the wells and change it for regular cell media. Collect the proteins from those wells at two, six, and 12 hours after changing the media. The induction of Cas9 protein was confirmed by its expression levels being similar amongst the transduced cells and the vehicle, notwithstanding the presence or absence of Shield-1.There is sufficient induction of Cas9 expression in the transduced A549 cell line two hours after treatment with Shield-1, which becomes negligible within six to 12 hours after withdrawal of Shield-1. Shield-1 treated transduced A549 cells decreased in number after 48 hours without affecting the Rinella sample, which was validated by immunoblotting using an antibody against the depleted RPA3 protein. Gene editing inversion or indel mutations were confirmed using surveyor nuclease assays.The lentivirus vector construct designed was a bicistronic system bearing another gene of interest under the same EF-1a promoter. A modified fluorescent protein, P2A, was added to trace infected cells. The expression of the mVenus protein was observed in the vehicle and A549 transduced cells, independent of DD-Cas9 expression and Shield-1 treatment.The efficient transduction of the cell line with DD-Cas9 vector is one of the rate limiting steps, for having healthy HEK293T cells and using their low passage number is crucial. The optimization of the ratio between DD-Cas9 plasmid, packaging plasmid, and envelope plasmid is very important for optimal transfection. But beside it, also the amount of Shield-1 that is needed to stabilize DD-Cas9 target cells is something that you need to be careful of.The DD-Cas9 with the Cre plasmid is also available, and it can be used to study the genetic interactions in in vivo studies based on Cre-lox system.

a-new-toolkit-for-evaluating-gene-functions-using-conditional-cas9

Research

JoVE Journal

Genetics

3.9K vistas.  Cold Spring Harbor Laboratory. Este método proporciona una edición rápida y controlada de genes CRISPR-Cas9 bajo una molécula pequeña, Shield-1. También ofrece menos efectos fuera del objetivo, menor toxicidad celular y también una mayor edición de genes controlada internamente cuando se compara con la activación constitutiva de Cas9. La principal ventaja de esta partícula es que se puede utilizar para la caracterización de genes esenciales, así como genes de supervivencia tumoral.La desestabilización d...