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

<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>

The authors declare no competing financial interests.

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 ...

pooled-crispr-based-genetic-screens-in-mammalian-cells

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JoVE Journal

Genetics

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Video: Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

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 ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

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

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

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

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

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

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

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers.

This protocol describes the steps needed to design and perform multiplexed targeting of enhancers with the deactivating fusion protein SID4X-dCas9-KRAB, also known as enhancer interference (Enhancer-i). This protocol enables the identification of enhancers that regulate gene expression and facilitates the dissection of relationships between enhancers regulating a common target gene.

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.