Name: pooled crispr-based genetic screens in mammalian cells
Uploaded: 2019-09-04T13:00:00
Description: Watch this Scientific Journal Video about Pooled CRISPR-Based Genetic Screens in Mammalian Cells at JoVE.com

This work was supported by Genome Canada, the Ontario Research Fund, and the Canadian Institutes for Health Research (MOP-142375, PJT-148802).

The experiments outlined below should&#160;follow&#160;the institute&#8217;s Environmental Health and Safety Office guidelines.
1. Pooled CRISPR sgRNA lentiviral library plasmid amplification
<ol>
	<li>Dilute the ready-made CRISPR sgRNA plasmid DNA library to 50 ng/&#956;L in TE (e.g., TKOv3).</li>
	<li>Electroporate the library using electrocompetent cells. Set up a total of four electroporation reactions as described below.
	<ol>
		<li>Add 2 &#956;L of 50 ng/&#956;L TKO library to 25 &#956...

<ol>
	<li>Jiang, F., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas9+Structures+and+Mechanisms.">CRISPR-Cas9 Structures and Mechanisms.</a> Annual Review of Biophysics. 46, 505-529 (2017).</li><li>Baliou, 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=CRISPR+therapeutic+tools+for+complex+genetic+disorders+and+cancer+(Review).">CRISPR therapeutic tools for complex genetic disorders and cancer (Review).</a> International Journal of Oncology. 53 (2), 443-468 (2018).</li><li>Hart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+CRISPR+Screens+Reveal+Fitness+Genes+and+Genotype-Specific+Cancer+Liabilities.">High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.</a> Cell. 163 (6), 1515-1526 (2015).</li><li>Morgens, D. W., Deans, R. M., Li, A., Bassik, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+CRISPR/Cas9+and+RNAi+screens+for+essential+genes.">Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes.</a> Nature Biotechnology. 34 (6), 634-636 (2016).</li><li>Evers, 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=CRISPR+knock-out+screening+outperforms+shRNA+and+CRISPRi+in+identifying+essential+genes.">CRISPR knock-out screening outperforms shRNA and CRISPRi in identifying essential genes.</a> Nature Biotechnology. 34 (6), 631-633 (2016).</li><li>Miles, L. A., Garippa, R. J., Poirier, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+execution,+and+analysis+of+pooled+in+vitro+CRISPR/Cas9+screens.">Design, execution, and analysis of pooled in vitro CRISPR/Cas9 screens.</a> The FEBS Journal. 283 (17), 3170-3180 (2016).</li><li>Wang, T., Wei, J. J., Sabatini, D. M., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+in+human+cells+using+the+CRISPR-Cas9+system.">Genetic screens in human cells using the CRISPR-Cas9 system.</a> Science. 343 (6166), 80-84 (2014).</li><li>Wang, 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=Identification+and+characterization+of+essential+genes+in+the+human+genome.">Identification and characterization of essential genes in the human genome.</a> Science. 350 (6264), 1096-1101 (2015).</li><li>Sanson, K. 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=Optimized+libraries+for+CRISPR-Cas9+genetic+screens+with+multiple+modalities.">Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities.</a> Nature Communications. 9 (1), 5416 (2018).</li><li>Hart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+Design+of+Genome-Wide+CRISPR/SpCas9+Knock-out+Screens.">Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knock-out Screens.</a> G3: Genes|Genomes|Genetics. 7 (8), 2719-2727 (2017).</li><li>Mair, B., Tomic, 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=Essential+gene+profiles+for+human+pluripotent+stem+cells+identify+uncharacterized+genes+and+substrate+dependencies.">Essential gene profiles for human pluripotent stem cells identify uncharacterized genes and substrate dependencies.</a> Cell Reports. 27 (2), 599-615 (2019).</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+knock-out+screening+in+human+cells.">Genome-scale CRISPR-Cas9 knock-out screening in human cells.</a> Science. 343 (6166), 84-87 (2014).</li><li>Sharma, S., Petsalaki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+CRISPR-Cas9+Based+Genome-Wide+Screening+Approaches+to+Study+Cellular+Signalling+Mechanisms.">Application of CRISPR-Cas9 Based Genome-Wide Screening Approaches to Study Cellular Signalling Mechanisms.</a> International Journal of Molecular Sciences. 19 (4), (2018).</li><li>Steinhart, Z., 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-wide+CRISPR+screens+reveal+a+Wnt-FZD5+signaling+circuit+as+a+druggable+vulnerability+of+RNF43-mutant+pancreatic+tumors.">Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors.</a> Nature Medicine. 23 (1), 60-68 (2017).</li><li>Wang, 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=Gene+Essentiality+Profiling+Reveals+Gene+Networks+and+Synthetic+Lethal+Interactions+with+Oncogenic+Ras.">Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras.</a> Cell. 168 (5), 890-903 (2017).</li><li>Zimmermann, 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=CRISPR+screens+identify+genomic+ribonucleotides+as+a+source+of+PARP-trapping+lesions.">CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.</a> Nature. 559 (7713), 285-289 (2018).</li><li>Deans, R. 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=Parallel+shRNA+and+CRISPR-Cas9+screens+enable+antiviral+drug+target+identification.">Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification.</a> Nature Chemical Biology. 12 (5), 361-366 (2016).</li><li>Trinh, T. J. J., Bloom, F., Hirsch, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STBL2:+an+Escherichia+coli+strain+for+the+stable+propagation+of+retroviral+clones+and+direct+repeat+sequences.">STBL2: an Escherichia coli strain for the stable propagation of retroviral clones and direct repeat sequences.</a> Focus. 16, 78-80 (1994).</li><li>Hart, T., Moffat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAGEL:+a+computational+framework+for+identifying+essential+genes+from+pooled+library+screens.">BAGEL: a computational framework for identifying essential genes from pooled library screens.</a> BMC Bioinformatics. 17, 164 (2016).</li><li>Wang, G. Z. 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=Identifying+drug-gene+interactions+from+CRISPR+knock-out+screens+with+drugZ.">Identifying drug-gene interactions from CRISPR knock-out screens with drugZ.</a> bioRxiv. , (2017).</li><li>Pedregosa, F. V., 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=Scikit-learn:+Machine+Learning+in+Python.">Scikit-learn: Machine Learning in Python.</a> Journal of Machine Learning Research. 12, 2825-2830 (2011).</li><li>Ketela, 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+comprehensive+platform+for+highly+multiplexed+mammalian+functional+genetic+screens.">A comprehensive platform for highly multiplexed mammalian functional genetic screens.</a> BMC Genomics. 12, 213 (2011).</li><li>Doench, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Am+I+ready+for+CRISPR?+A+user's+guide+to+genetic+screens.">Am I ready for CRISPR? A user's guide to genetic screens.</a> Nature Review Genetics. 19 (2), 67-80 (2018).</li><li>Hartenian, E., Doench, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+and+functional+genomics+using+CRISPR/Cas9+technology.">Genetic screens and functional genomics using CRISPR/Cas9 technology.</a> FEBS Journal. 282 (8), 1383-1393 (2015).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAGeCK+enables+robust+identification+of+essential+genes+from+genome-scale+CRISPR/Cas9+knock-out+screens.">MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knock-out screens.</a> Genome Biology. 15 (12), 554 (2014).</li><li>Sheel, A., Xue, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Amplifications+Cause+False+Positives+in+CRISPR+Screens.">Genomic Amplifications Cause False Positives in CRISPR Screens.</a> Cancer Discovery. 6 (8), 824-826 (2016).</li><li>Meyers, R. 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=Computational+correction+of+copy+number+effect+improves+specificity+of+CRISPR-Cas9+essentiality+screens+in+cancer+cells.">Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells.</a> Nature Genetics. 49 (12), 1779-1784 (2017).</li><li>Henser-Brownhill, T., Monserrat, J., Scaffidi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+an+arrayed+CRISPR-Cas9+library+targeting+epigenetic+regulators:+from+high-content+screens+to+in+vivo+assays.">Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays.</a> Epigenetics. 12 (12), 1065-1075 (2017).</li></ol>

Due to its simplicity of use and high pliability, CRISPR technology has been widely adopted as the tool of choice for precise genome editing. Pooled CRISPR screening provides a method to interrogate thousands of genetic perturbations in a single experiment. In pooled screens, sgRNA libraries serve as molecular barcodes, as each sequence is unique and is mapped to the targeted gene. By isolating the genomic DNA from the cell population, genes causing the phenotype of interest can be determined by quantifying sgRNA abundan...

CRISPR-Cas, short for clustered regularly interspaced short palindromic repeats and CRISPR-associated nuclease, consists of a single nuclease protein (e.g., Cas9) in complex with a synthetic guide RNA (sgRNA). This ribonucleoprotein complex targets the Cas9 enzyme to induce double-stranded DNA breaks at a specific genomic locus1. Double-stranded breaks can be repaired via homology directed repair (HDR) or, more commonly, through non-homologous end joining (NHEJ), an error prone repair mechanism that results in insertion and/or deletions (INDELS) that frequently disrupt gene function1. The efficiency and simplicity of CRISPR enables a previously unattainable level of genomic targeting that far surpasses previous genome editing technologies [i.e., zinc finger nucleases (ZNF) or transcription activator-like effector nucleases (TALENS), both of which suffer from heightened design complexity, lower transfection efficiency, and limitations in multiplex gene editing2].
The basic research application of CRISPR single-guide RNA-based genome editing has allowed scientists to efficiently and inexpensively interrogate the functions of individual genes and topology of genetic interaction networks. The ability to perform functional genome-wide screens has been greatly enhanced by use of the CRISPR-Cas system, particularly when compared to earlier genetic perturbation technologies such as RNA interference (RNAi) and gene trap mutagenesis. In particular, RNAi suffers from high off-target effects and incomplete knockdown, resulting in lower sensitivity and specificity compared to CRISPR3,4,5, while gene trap methods are only feasible in haploid cells for loss-of-function screens, limiting the scope of cell models that can be interrogated6. The ability of CRISPR to generate complete gene knock-out provides a more biologically robust system to interrogate mutant phenotypes, with low noise, minimal off-target effects and consistent activity across reagents5. CRISPR-Cas9 sgRNA libraries that target the entire human genome are now widely available, allowing simultaneous generation of thousands of gene knock-outs in a single experiment3,7,8,9.
We have developed unique CRISPR-Cas9 genome-wide sgRNA lentiviral libraries called the Toronto Knock-out (TKO) libraries (available through Addgene) that are compact and sequence-optimized to facilitate high resolution functional genomics screens. The latest library, TKOv3, targets ~18,000 human protein-coding genes with 71,090 guides optimized for editing efficiency using empirical data10. Additionally, TKOv3 is available as a one-component library (LCV2::TKOv3, Addgene ID #90294) expressing Cas9 and sgRNAs on a single vector, alleviating the need to generate stable Cas9-expressing cells, enabling genome-wide knock-out across a broad range of mammalian cell types. TKOv3 is also available in a vector without Cas9 (pLCKO2::TKOv3, Addgene ID# 125517) and can be utilized in cells that express Cas911.
A genome-wide CRISPR-Cas9 edited cell population can be exposed to different growth conditions, with the abundance of sgRNAs over time quantified by next-generation sequencing, providing a readout to assess drop-out or enrichment of cells with traceable genetic perturbations. CRISPR knock-out libraries can be harnessed to identify genes that, upon perturbation, cause cellular fitness defects, moderate drug sensitivity (e.g., sensitive or resistant genes), regulate protein expression (e.g., reporter), or are required for a certain pathway function and cellular state12,13,14. For example, differential fitness screens in a cancer cell line can identify both depletion or reduction of oncogenes and enrichment or an increase of tumor suppressors genes3,14,15. Similarly, using intermediate doses of therapeutic drugs can reveal both drug resistance and sensitization genes16,17.
Provided here is a detailed screening protocol for genome-scale CRISPR-Cas9 loss-of-function screening using the Toronto Knock-out libraries (TKOv1 or v3) in mammalian cells from library generation, screening performance to data analysis. Although this protocol has been optimized for screening using the Toronto Knock-out libraries, it can be applied and become scalable to all CRISPR sgRNA pooled libraries.

<table>
	<tbody>
		<tr>
			<td>0.22 micron filter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30&#176;C plate incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C shaking incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C, 5% CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>Promega</td>
			<td>V4221</td>
			<td></td>
		</tr>
		<tr>
			<td>50X TAE buffer</td>
			<td>BioShop</td>
			<td>TAE222.4</td>
			<td></td>
		</tr>
		<tr>
			<td>6 N Hydrochloric acid solution</td>
			<td>BioShop</td>
			<td>HCL666.500</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alamar blue</td>
			<td>ThermoFisher Scientific</td>
			<td>DAL1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue-light transilluminator</td>
			<td>ThermoFisher Scientific</td>
			<td>G6600</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin,Heat Shock Isolation, Fraction V. Min. 98%, Biotechnology grade</td>
			<td>Bioshop</td>
			<td>ALB001.250</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagles Medium</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td>Cel culture media</td>
		</tr>
		<tr>
			<td>Electroporation cuvettes</td>
			<td>BTX</td>
			<td>45-0134</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>BTX</td>
			<td>45-0651</td>
			<td></td>
		</tr>
		<tr>
			<td>Endura electrocompetent cells</td>
			<td>Lucigen</td>
			<td>90293</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T packaging cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>recommend passage number &#60;15</td>
		</tr>
		<tr>
			<td>Hexadimethrine Bromide (Polybrene)</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>Cationic polymer to enhance transduction efficiency</td>
		</tr>
		<tr>
			<td>Hexadimethrine Bromide (Polybrene)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar plates with carbenicillin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB medium with carbenicillin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low molecular weight DNA ladder</td>
			<td>New England Biolabs</td>
			<td>N3233S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Ultra II Q5 Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0544L</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Reduced serum media</td>
		</tr>
		<tr>
			<td>Plasmid maxi purification kit</td>
			<td>Qiagen</td>
			<td>12963</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G (envelope plasmid)</td>
			<td>Addgene</td>
			<td>Plasmid #12259</td>
			<td>lentiviral system</td>
		</tr>
		<tr>
			<td>psPAX2 (packaging plasmid)</td>
			<td>Addgene</td>
			<td>Plasmid #12260</td>
			<td>lentiviral system</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Wisent</td>
			<td>400-160-UG</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick gel extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Q32853</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit fluorometer</td>
			<td>ThermoFisher Scientific</td>
			<td>Q33226</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse A</td>
			<td>Invitrogen</td>
			<td>12091021</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C recovery medium</td>
			<td>Invitrogen</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>SYRB Safe DNA gel stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>Toronto KnockOut CRIPSR library (TKOv3) - Cas9 included</td>
			<td>Addgene</td>
			<td>Addgene ID #90203</td>
			<td>Genome-wide CRISPR library , includes Cas9, 71,090 sgRNA</td>
		</tr>
		<tr>
			<td>Toronto KnockOut CRIPSR library (TKOv3) - non-cas9</td>
			<td>Addgene</td>
			<td>Addgene ID #125517</td>
			<td>Genome-wide CRISPR library, non-Cas9, 71,090 sgRNA</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) solution, pH8.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>16500500</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard genomic DNA purification kit</td>
			<td>Promega</td>
			<td>A1120</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE 9 DNA transfection reagent</td>
			<td>Roche</td>
			<td>06 365 809 001</td>
			<td>Lipid based transfection reagent</td>
		</tr>
	</tbody>
</table>

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.

Overview of genome-scale CRISPR screening workflow
Figure 1 illustrates an overview of the pooled CRISPR screening work flow, starting with infection of target cells with CRISPR library lentivirus at a low MOI to ensure single integration events and adequate library representation (typically 200- to 1000-fold). Following infection, cells are treated with the antibiotic puromycin to ...

Watch this Scientific Journal Video about Pooled CRISPR-Based Genetic Screens in Mammalian Cells at JoVE.com

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

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 ...

CRISPR screened drop-out screens provide researchers with a simple, efficient, and inexpensive method to interrogate chain function on the genome-wide level. The advantage of CRISPR is its pliability to edit any gene by simply changing the guide sequence. CRISPR guide libraries enable researchers to interrogate the entire genome of any organism in one experiment in an unbiased, systematic way.Currently, CRISPR screens are being used to identify essential genes across hundreds of human cancers and to map genetic interactions. Screens can also comprehensively profile drugs to reveal drug mechanisms of action. The CRISPR libraries described here target human cells.However, guide libraries targeting other species, such as mouse, are available and can be screened similarly. Performing genome-wide screens in human cells can be daunting in practice, as it involves the handling of tens of millions in cells and requires analysis of large sets of data. Before starting a screen, ensure cell lines are carefully characterized.This includes knowing the purity of your cell line, doubling time, lentivirus transduction efficiencies, and sensitivities to antibiotic selection agents. A visual demonstration will provide a picture of how to practically handle the millions of cells required in a screen and keep track of thousands of perturbations in a systematic manner. Demonstrating the procedure will be Andrea Habsid, Kamaldeep Aulakh, and Ryan Climie, all technicians from my laboratory.Begin by transforming the ready-made CRISPR sgRNA plasmid into electrocompetent cells and growing them according to manuscript directions. When ready to harvest the colonies, add seven milliliters of LB with carbenicillin to each plate and scrape the colonies off with the cell spreader. Use a 10-milliliter pipette to transfer the scraped cells into a sterile one-liter conical flask, and rinse the plate with five milliliters of LB with carbenicillin.Again, transfer the rinsing solution to the flask. Centrifuge the cells according to manuscript directions, and then, determine the weight of the wet pellet. Purify the plasmid DNA using a Maxi or Mega scale plasmid purification kit.Prepare cells for transfection by seeding 293 T-cells and incubating them overnight. On the next day, prepare the three transfection plasmid mixture for 15-centimeter plates. Calculate the amount of plasmid needed for one transfection and make a mix of plasmids for the number of plates, plus one, to be transfected.Next, prepare a lipid-based transfection reagent for each transfection and aliquot-reduced serum media into individual 1.5-milliliter microcentrifuge tubes for the number of plates to be transfected. Add the transfection reagent, mix gently, and incubate at room temperature for five minutes. After the incubation, add DNA to the transfection reagent at a three to one ratio of transfection reagent to micrograms of DNA complex.Mix the solution gently and leave at room temperature for 30 minutes. Add the transfection mixture to packaging cells and incubate them according to manuscript directions. On the day of viral harvest, check the cells for abnormal and fused morphology as an indication of good virus production, and harvest the lentivirus by collecting the supernatant and transferring it to a sterile centrifuge tube.Begin by selecting the CRISPR guide RNA library coverage to be maintained throughout the screen. Based on the library coverage, determine the number of cells required to maintain it per guide RNA and the number of cells required for infection at MOI 0.3. Next, determine the number of plates required to set up the infection and then, harvest and seed the cells to each plate.Add hexadimethrine bromide to all plates and add the required volume of virus to screening and control two plates. Do not add virus to control one. Replace that volume with media.Mix the plates thoroughly by tilting and place the plates into an incubator, making sure that they are level. Harvest infected cells according to manuscript directions and collect three replicates of cell pellets from the pooled cells for genomic DNA extraction. Centrifuge the cells at 500 times g for five minutes and wash them with PBS.Label the tubes and freeze dry the cell pellets at minus 80 degrees Celsius. Split the pool of infected cells to three replicate groups, making sure to maintain library coverage within each replicate. Seed the cells at a density that would normally be used when expanding them.Use the same number of cells for each replicate and the same total number of cells between replicates. Continue to passage the cells and harvest three replicates of cell pellets from each replicate of pooled infected cells for up to 15 to 20 cell doublings. At each passage, harvest the cells from all plates in each replicate with each other.Label each pellet with the time and replicate designation. Set up PCR1 according to manuscript directions with a total of 100 micrograms of genomic DNA at 3.5 micrograms of genomic DNA per 50-microliter reaction and set up identical 50-microliter reactions to achieve desired coverage. Set up one PCR tube reaction according to manuscript directions.Use five microliters of the pooled PCR1 product as a template and use unique index primer combinations for each individual sample to allow pooling of sequencing library samples. After completing the PCR, run the PCR tube product on a 2%agarose gel at low voltage for one to 1 1/2 hours. Visualize the product on a blue light transilluminator and excise the 200 base pair band.Purify the DNA and measure its quantity and quality with both a spectrophotometer and a fluorometer. An ideal guide RNA library should have every single guide RNA represented at similar quantities. Next generation sequencing can be used to confirm that the library has a tight distribution of guide RNAs.Precision-recall analysis can be used to evaluate screen performance. A high-performing screen should recover a large number of essential genes at a base factor larger than six and a false discovery rate of less than 5%The precision-recall curve should have a sharp elbow and a straight line to the terminal point. The Bayes factor represents a confidence measure that the gene knockout results in a fitness defect.High scores indicate increased confidence, while low scores suggest that the gene knockout provides growth advantages. Genome-wide knockout pools can be cultured in the presence of excess drug agent to look for suppressor resistance genes. To perform a positive selection screen for suppressor of thymidine block, normalized read counts for all guide RNAs at T0 are plotted against mean-normalized read counts for thymidine-treated samples.A key detail to successfully performing CRISPR screens is maintaining good distribution of each guide RNA, starting from transfection of the plasmid to transduction of cells. This will minimize the chance of random effects that can skew guide RNA representation and lead to false positive or negative results. Following a screen validation, experiments should be done to confirm hits because the primary screen only identifies potential hits.Depending on the biology of the hits, various methods can be used. CRISPR editing, combined with next-generation sequencing, has enabled genome-scale loss of function screens in diverse human model systems. Due to the simplicity of CRISPR, these types of screens are now broadly accessible to all researchers, allowing more scientists to utilize functional genetic methods to study biological processes and disease.

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