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>
2. Small guide RNA (sgRNA) design
<ol>
	<li>Design small guide RNA (sgRNA) that will be cloned into the DD-Cas9 vector by using one of several algorithms, targeting a specific genomic region. 
	NOTE: To reduce the off-target effects, select sgRNA sequences with the highest score by using the website: http://crispr.mit.edu.</li>
	<li>Design and order two oligonucleotides per sgRNA sequence to make a complete sgRNA. 
	&#8203;NOTE: Since the plasmid will be digested by the BsmBI enzyme, design the forward and the reverse oligonucleotide by adding BsmBI digestion overhangs to the sgRNA sequence. Use the template from Table 1.</li>
</ol>
3. Cloning of sgRNA into the lentiviral DD-Cas9 vector
<ol>
	<li>Dephosphorylate the 5&#8242;-ends of DD-Cas9 plasmid by using alkaline phosphatase (FastAP) in a restriction enzyme digestion reaction. 
	NOTE: Vectors can re-circularize during the ligation especially when they are linearized by a single restriction enzyme. To ensure the vector will not re-circularize, de-phosphorylate the plasmid by adding phosphatase directly into the digestion reaction.
	<ol>
		<li>Dephosphorylate and digest the plasmid with BsmBI enzyme by preparing a mix of 5 &#181;g of DD-Cas9 plasmid, 6 &#181;L of digest buffer (10x), 0.6 &#181;L of DTT (100 mM), 3 &#181;L of BsmBI, 3 &#181;L of FastAP, add up to 60 &#181;L nuclease-free water to make 60 &#181;L the total reaction volume. 
		NOTE: Add the BsmBI enzyme and FastAP to the mixture in the end.</li>
		<li>Incubate the mixture for 30 min at 37&#160;&#176;C heat block or thermocycler.</li>
	</ol>
	</li>
	<li>Purify the digested DD-Cas9 plasmid.
	<ol>
		<li>Prepare an 0.8% DNA agarose gel to remove incompletely digested plasmid and traces of enzymes. Use a wider gel-comb for better separation and run the gel at 90 V.</li>
		<li>Visualize bands under 360-365 nm UV light and cut the larger plasmid band out of the gel. Discard the 2 kb fragment which is corresponding to the filler.</li>
		<li>Purify the large cut gel-band by using a commercial gel purification kit and follow the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Ligate annealed oligos with digested DD-Cas9 plasmid.
	<ol>
		<li>Phosphorylate and anneal the sgRNA oligos.
		<ol>
			<li>Prepare the mix of 1 &#181;L of T4 Ligation Buffer (10x), 1 &#181;L of Oligo 1 (100 &#181;M), 1 &#181;L of Oligo 2 (100 &#181;M), 6.5 &#181;L of nuclease-free water. Lastly, add 0.5 &#181;L of T4 PNK. The total volume of this reaction is 10 &#181;L.</li>
			<li>Add the reaction-mix to the thermocycler with the following conditions: 37&#160;&#176;C for 30 min, 95 &#176;C for 5 min, and then ramp down to 25 &#176;C with the speed of 5 &#176;C/min. 
			&#8203;NOTE: The PNK must be heat-inactivated before the oligonucleotides are put into the ligation otherwise the PNK will phosphorylate the vector. The phosphorylation step and annealing step are performed together in a thermocycler. The phosphorylation step where 5&#39; phosphate is added to the sgRNA oligonucleotides is required for the ligation to occur.</li>
		</ol>
		</li>
		<li>Ligate DD-Cas9 digested plasmid and sgRNA oligos.
		<ol>
			<li>Dilute the annealed oligos to 1:200 in nuclease-free water and use 50 ng of plasmid DNA in the ligation reaction. 
			&#8203;NOTE: Use the molar ratio of the 6 insert : 1 vector, to promote insert integration or use a ligation calculator to calculate the molar ratio: http://nebiocalculator.neb.com/#!/ligation.</li>
			<li>Prepare the ligation reaction mix: 50 ng of digested and purified DD-Cas9 vector, appropriate volume of diluted annealed oligos, 1 &#181;L of T4 Ligation Buffer (10x), 1 &#181;L of T4 DNA Ligase, up to 10 &#181;L nuclease-free water to obtain the total volume of the reaction 10 &#181;L.</li>
			<li>If using &#34;high concentration&#34; ligase, incubate the reaction at room temperature for 5 min. Otherwise, incubate at room temperature for 2 h, or to achieve a higher yield of ligation, incubate at 16 &#176;C overnight. 
			&#8203;NOTE: Heat-inactivate if using the standard T4 DNA Ligase at 65 &#176;C for 20 minutes. Do not heat-inactivate if you are using ligase master mixes.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Bacterial transformation
<ol>
	<li>Pre-warm 10 cm LB-ampicillin agar plates at room temperature.</li>
	<li>Thaw 50 &#181;L of &#34;One shot Stbl3&#34; competent bacterial cells on ice.</li>
	<li>Add 2-3 &#181;L of ligation mixture to 50 &#181;L of competent bacterial cells and gently mix by flicking the bottom of the tube with a finger 4 times. Incubate on ice for 30 min.</li>
	<li>Heat-shock the cells at exactly 42 &#176;C for 40 seconds, using a timer. Cool down the bacterial cells on ice for 5 min.</li>
	<li>Add 500 &#181;L of SOC media without antibiotic to the bacterial cells and grow them in the shaking incubator at 250 rpm for 45 min, at 37 &#176;C.</li>
	<li>Spread 100 &#181;L of transformed bacteria on pre-warm LB-ampicillin plates and incubate overnight at 37 &#176;C.</li>
</ol>
5. Mini/maxi-prep of ligated plasmid
<ol>
	<li>Prepare a maxi/mini-prep starter culture.
	<ol>
		<li>The next day pick a single bacterial clone and inoculate a starter culture with 8 mL of LB media containing ampicillin.</li>
		<li>Grow the bacteria in the shaking incubator at 250 rpm, 37 &#176;C for 10-12 h.</li>
		<li>Use 3-5 mL of bacteria for mini-prep or use it as a starter culture for maxi-prep. Use the rest of the bacteria as a bacterial glycerol-stock by adding 100% glycerol in the ratio of bacterial culture:glycerol is 1:1. 
		&#8203;NOTE: Here, we will describe the mini-prep procedure.</li>
	</ol>
	</li>
	<li>Mini-prep procedure
	<ol>
		<li>Harvest 3-5 mL of bacterial cells and centrifuge at 12,000 x g for 1 min.</li>
		<li>Resuspend the pellet of bacterial cells with 200 &#181;L of P1 buffer, containing RNase A and stored at 4 &#176;C.</li>
		<li>Add 200 &#181;L of buffer P2 and mix gently by inverting the tube 10 times until the liquid clarifies.</li>
		<li>Add 300 &#181;L of buffer P3 and mix by inverting the tube 10 times. The liquid will become cloudy. Proceed immediately with centrifuging the samples at 12,000 x g for 10 min.</li>
		<li>Transfer clear supernatant to the spin column and centrifuge at 12,000 x g for 1 min. 
		Discard the flow-through. 
		&#8203;NOTE: Make sure the supernatant is clarified. Particles can clog the spin column and reduce the column efficacy.</li>
		<li>Add 400 &#181;L of PD buffer and centrifuge at 12,000 x g for 1 min. Discard the flow-through.</li>
		<li>Add 600 &#181;L of PW buffer to wash the spin column and centrifuge at 12,000 x g for 1 min. Discard the flow-through.</li>
		<li>Centrifuge again the spin column at top speed for 3 min to remove the buffer residues. Discard the flow-through and let it sit for 2 min.</li>
		<li>Place the spin column to a fresh tube and add 50 &#181;L of elution buffer. Incubate for 5 min and centrifuge at top speed for 2 min.</li>
		<li>Use the nanodrop device to measure the concentration of purified DD-Cas9 plasmid with the sgRNA insert (ng/&#181;L).</li>
	</ol>
	</li>
	<li>Validate the sgRNA cloning by DNA sequencing of the DD-Cas9 plasmid. Use the U6 primer sequence in Table 2.</li>
</ol>
6. Lentiviral preparation
<ol>
	<li>Prepare HEK293T packaging cells for transfection.
	<ol>
		<li>Use healthy HEK293T up to passage 10 and seed them evenly at density 1-2 x 106 cells per 10 cm tissue culture plate. Use 10&#160;mL of Glucose Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% filtered Fetal Bovine Serum (FBS). Incubate cells in the tissue culture incubator, at 5% CO2 and 37 &#176;C for 20 h.</li>
		<li>Next day, confirm that cells reached 70% confluency by brightfield microscopy and that they are evenly dispersed across the plate. Change media to cells 1 h before the transfection with the final volume of 10 mL. 
		&#8203;NOTE: The media should be absent of antibiotics and antimycotics.</li>
		<li>Prepare the mixture of 2 tubes with 500 &#181;L of warm media (e.g., OptiMEM).
		<ol>
			<li>Add 25 &#181;L of transfection reagent to tube 1 containing warm 500 &#181;L of media, and incubate at room temperature for 5 min.</li>
			<li>Meanwhile prepare&#160;a mix of plasmids to tube 2 containing 3.5 &#181;g of DD-Cas9 containing cloned sgRNA, 6 &#181;g of the packaging plasmid (psPAX2), and 3 &#181;g of the envelope plasmid (pMD2.G) into warm 500 &#181;L of media.</li>
			<li>Mix tube 1 with tube 2 to form a transfection mixture and incubate at room temperature for 20 min.</li>
			<li>Dropwise the transfection mixture to HEK293T cells and incubate plate in the tissue culture incubator, at 5% CO2 and 37 &#176;C overnight.</li>
			<li>After 18 h, carefully change the media to 10 mL of fresh DMEM with 10% FBS to remove the transfection reagent. Incubate for the next 48 h.</li>
			<li>After 48 h, collect the supernatant with a 10 mL syringe and pass it through a 0.45 &#181;m filter.</li>
			<li>Aliquot and store the virus supernatant at -80 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Determining virus titer and transduction efficacy with flow cytometry
<ol>
	<li>Seed the HEK293T cells with 5 x 105 cells/well into a 6-well plate. Seed cells into two 6-well plates. Use one plate for counting the next day.</li>
	<li>Incubate the plates in the incubator at 37 &#176;C, 95% humidity, 5% CO2 overnight to reach 50-60% confluency the next day.</li>
	<li>The next day, use one of the 6-well plates to count the cells in 6 wells.</li>
	<li>Thaw the virus aliquot that will be used to perform the serial dilution and add 8 &#181;g/mL of polybrene reagent.</li>
	<li>Prepare DMEM with 10% FBS for serial dilution of the virus and add 8 &#181;g/mL of polybrene reagent.</li>
	<li>Prepare 2 mL of ten-fold serial dilutions of the lentivirus from 1 x 10-1 to 1 x 10-4 in polybrene-containing media.</li>
	<li>Remove media from 6-well plate and add 1 mL of viral dilutions to wells. Leave one well with media alone as negative control and one well with 100% virus.</li>
	<li>Incubate cells with the virus in the incubator for 24 h.</li>
	<li>The next day, remove the media with virus from 6-well plates and change it for 2 mL of fresh DMEM with 10% FBS. Incubate cells for 48-72 h. Observe GFP by using a fluorescent microscope every day.</li>
	<li>After 48-72 h, detach the cells and resuspend them in MACS buffer.</li>
	<li>Use a flow cytometer to determine the percentage of GFP expression.</li>
	<li>Calculate virus titer by using the following formula: TU/mL = (Number of cells transduced x Percent fluorescent x Dilution Factor)/(Transduction Volume in mL)</li>
</ol>
8. Lentiviral transduction of target cells
<ol>
	<li>Plate the cell line of interest to a 10 cm plate and incubate overnight to reach confluency 50-60% the next day. 
	NOTE: We used the A549 cell line expressing DD-Cas9 and two independent RPA3-gene sgRNAs. We also used the A549 cell line expressing vector with Renilla control. We seeded those cells at the density of 2 x 103cells/cm2 and incubated them at 37 &#176;C, 95% humidity, 5% CO2 overnight to reach 50-60% confluency the next day. We used RPMI media with 10% filtered FBS. We proceeded with the next steps as follow:</li>
	<li>The next day, infect the cells with 500-2000 &#181;L of virus particles in 10 mL total volume of culture medium. Incubate viral media for 24 h.</li>
	<li>The next day, change the viral media to culture media and determine the percentage of GFP positive cells by using flow cytometry.</li>
	<li>Select GFP positive cells by FACS cell sorting or by using bleomycin selection.</li>
	<li>Expand positively selected cells and freeze a stock.</li>
</ol>
9. Conditional induction of Cas9 mediated gene editing
<ol>
	<li>Plate positively selected cells 24 h after infection as well as untransduced cells to 12-well plate separately. Wait until the cells attach and change the media to cell culture media containing 200 nM of Shield-1. Replace the media in the plates with transduced and untransduced-cells, leaving 2 wells per plate with regular media as a negative control.</li>
	<li>Incubate and extract proteins from each well at different time points: time 0, 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h after adding the Shield-1 to the wells.</li>
	<li>Then remove the media with Shield-1 from the rest of the wells and change it for regular cell media.</li>
	<li>Collect the proteins from those wells 2 h, 6 h, and 12 h after changing the media.</li>
	<li>Visualize by western blot analysis the reversibility and rapidity of destabilized DD-Cas9 protein regulation after the addition and the withdrawal of its ligand, Shield-1. Use antibody directed towards DYKDDDDK Tag to visualize proteins and a control antibody targeting for example beta-tubulin.</li>
	<li>Determine the optimal dose of Shield-1 by dose-response curve observed by Western Blot analysis.</li>
</ol>
10. Validation of gene editing
NOTE: The GFP expression assays, such as flow cytometry analysis and bleomycin selection marker only confirm successful CRISPR reagent delivery but they do not determine if the desired sequence was successfully targeted. The most common assays to confirm successful gene targeting by the CRISPR experiment are Sanger DNA sequencing, Next-generation sequencing, the Surveyor Nuclease Assay, the Tracking of Indels by Decomposition (TIDE) Assay, or western blot analysis16,17,18.
<ol>
	<li>Plate positively selected cells and change media to 200 nM of Shield-1 containing media. Incubate cells for 5 days, changing media with Shield-1 every 3 days.</li>
	<li>Use the above-mentioned validation techniques 5 days after DD-Cas9 induction by Shield-1.</li>
</ol>

<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 authors declare that they have no conflict of interest.

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><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 &amp;amp; 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>&amp;alpha;-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...

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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 ...

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Research

JoVE Journal

Genetics

4.1K Views.  Cold Spring Harbor Laboratory. Deze methode biedt snelle, temperatuurgecontroleerde CRISPR-Cas9 genbewerking onder een klein molecuul, Shield-1. Het biedt ook minder off-target effecten, lagere celtoxiciteit, en ook hogere intern gecontroleerde genbewerking bij het vergelijken met constitutieve activering van Cas9. Het belangrijkste voordeel van dit deeltje is dat het kan worden gebruikt voor karakterisering van essentiële genen, evenals tumoroverlevingsgenen.Destabilisatie van DD-Cas9 is onafhankelijk van de expre...

Video: A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

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

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

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

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

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

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

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

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

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

Cancer Research

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing&#160;in less than two weeks.

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The goal of this protocol is to enable seamless CRISPR/Cas9 editing of the C. elegans genome using assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. This protocol can be used to make a variety of genetic modifications in C. elegans including insertions, deletions, gene replacements and codon substitutions.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been ...

Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.

Here, we present a protocol that integrates CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to identify candidate genes that are crucial for the abnormal proliferative capacity of hematopoietic stem and progenitor cells.

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. ...

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

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