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Genetic screens in human cells using the CRISPR-Cas9 system. ​

Literature Information ​

DOI10.1126/science.1246981
PMID24336569
JournalScience (New York, N.Y.)
Impact Factor45.8
JCR QuartileQ1
Publication Year2014
Times Cited1552
KeywordsCRISPR-Cas9, genetic screening, human cells, sgRNA, genome editing
Literature TypeEvaluation Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.
ISSN0036-8075
Pages80-4
Issue343(6166)
AuthorsTim Wang, Jenny J Wei, David M Sabatini, Eric S Lander

TL;DR ​

This study presents a pooled, loss-of-function genetic screening method utilizing a genome-scale lentiviral sgRNA library to explore essential genes and drug resistance mechanisms in human cell lines through CRISPR-Cas9 technology. The findings highlight the effectiveness of sgRNA for generating knockout collections and reveal associations between sgRNA efficiency and specific sequence motifs, underscoring the potential of this approach for systematic genetic analysis in mammalian cells.

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CRISPR-Cas9 · genetic screening · human cells · sgRNA · genome editing

Abstract ​

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

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Primary Questions Addressed ​

  1. What are the advantages of using pooled, loss-of-function genetic screening approaches over traditional methods in mammalian genetics?
  2. How does the integration of sgRNA expression cassettes into the genome improve the tracking of mutant pools during screening?
  3. In what ways can the identification of specific sequence motifs associated with sgRNA efficiency influence future CRISPR-Cas9 experiments?
  4. What implications do the findings from the resistance screen to 6-thioguanine have for understanding the DNA mismatch repair pathway?
  5. How can the results of the negative selection screen for essential genes inform the development of targeted therapies in cancer treatment?

Key Findings ​

Research Background and Objectives ​

The study investigates the use of the CRISPR-Cas9 system for conducting large-scale genetic screens in human cells. Traditional methods for loss-of-function genetic screening in mammalian cells have been limited, primarily due to challenges in effectively inactivating both alleles of a gene. The objective of this research is to establish a robust and efficient method for performing genetic screens using a pooled library of single-guide RNAs (sgRNAs) that can facilitate both positive and negative selection.

Main Methods/Materials/Experimental Design ​

The research employs a genome-scale lentiviral sgRNA library, consisting of 73,000 sgRNAs targeting various genes, which are stably integrated into the genome of human cell lines. The study is structured around several key experimental components:

  1. sgRNA Library Construction: The library is designed to include multiple sgRNAs per gene, with a focus on coding exons and filtered for potential off-target effects.

  2. Cell Line Preparation: Two human cell lines, KBM7 (near-haploid) and HL60 (pseudo-diploid), are used. A Cas9 nuclease is expressed in these cells under a doxycycline-inducible promoter.

  3. Pooled Screening Approach: Cells are infected with the lentiviral sgRNA library at a low multiplicity of infection (MOI). Following selection under specific conditions (e.g., treatment with 6-thioguanine or etoposide), the abundance of sgRNAs in the surviving cell population is analyzed through high-throughput sequencing.

  4. Data Analysis: The abundance of sgRNAs is monitored before and after selection, allowing for the identification of genes that confer resistance or are essential for cell viability.

Mermaid diagram

Key Results and Findings ​

  1. Successful Knockout Generation: The CRISPR-Cas9 system effectively induced mutations at targeted loci, resulting in loss-of-function alleles in the cell lines.

  2. Identification of Essential Genes: In a screen for resistance to 6-thioguanine, sgRNAs targeting known components of the DNA mismatch repair pathway were significantly enriched, validating the screening approach.

  3. Discovery of Novel Genes: In a screen for resistance to etoposide, unexpected genes such as CDK6 were identified alongside the expected TOP2A, indicating the utility of this method in discovering new gene functions.

  4. sgRNA Efficacy Insights: The study reveals that sgRNA efficiency correlates with specific sequence motifs, which can be used to predict more effective sgRNAs.

Main Conclusions/Significance/Innovativeness ​

The research demonstrates that the CRISPR-Cas9 system is a powerful tool for systematic genetic analysis in mammalian cells. It offers significant advantages over previous methods, including the ability to achieve complete gene inactivation, reduced off-target effects, and high coverage of target genes. The findings pave the way for more comprehensive functional genomics studies and could enhance our understanding of gene function in various biological contexts.

Research Limitations and Future Directions ​

  1. Limitations: The study is primarily focused on proliferative phenotypes and may not address the full range of biological processes. Additionally, while off-target effects were minimal, they still pose a potential risk in broader applications.

  2. Future Directions: Future research could expand the range of biological processes studied using this method. There is also potential for developing improved sgRNA libraries based on the insights gained regarding sgRNA efficacy and specificity.

Summary Table of Key Findings ​

AspectDetails
MethodologyCRISPR-Cas9 with a genome-scale sgRNA library
Cell Lines UsedKBM7 and HL60
Key Findings- Identification of essential genes
- Discovery of novel genes involved in drug resistance
ConclusionsCRISPR-Cas9 is effective for large-scale genetic screens
LimitationsFocus on proliferative phenotypes; potential off-target effects
Future DirectionsBroaden application to various biological processes

References ​

  1. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. - Vikram Pattanayak;Steven Lin;John P Guilinger;Enbo Ma;Jennifer A Doudna;David R Liu - Nature biotechnology (2013)
  2. Efficient genome editing in zebrafish using a CRISPR-Cas system. - Woong Y Hwang;Yanfang Fu;Deepak Reyon;Morgan L Maeder;Shengdar Q Tsai;Jeffry D Sander;Randall T Peterson;J-R Joanna Yeh;J Keith Joung - Nature biotechnology (2013)
  3. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. - Haoyi Wang;Hui Yang;Chikdu S Shivalila;Meelad M Dawlaty;Albert W Cheng;Feng Zhang;Rudolf Jaenisch - Cell (2013)
  4. Eukaryotic DNA mismatch repair. - R D Kolodner;G T Marsischky - Current opinion in genetics & development (1999)
  5. Topoisomerase levels determine chemotherapy response in vitro and in vivo. - Darren J Burgess;Jason Doles;Lars Zender;Wen Xue;Beicong Ma;W Richard McCombie;Gregory J Hannon;Scott W Lowe;Michael T Hemann - Proceedings of the National Academy of Sciences of the United States of America (2008)
  6. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. - Jason Moffat;Dorre A Grueneberg;Xiaoping Yang;So Young Kim;Angela M Kloepfer;Gregory Hinkle;Bruno Piqani;Thomas M Eisenhaure;Biao Luo;Jennifer K Grenier;Anne E Carpenter;Shi Yin Foo;Sheila A Stewart;Brent R Stockwell;Nir Hacohen;William C Hahn;Eric S Lander;David M Sabatini;David E Root - Cell (2006)
  7. Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells. - Ge Guo;Wei Wang;Allan Bradley - Nature (2004)
  8. A loss-of-function RNA interference screen for molecular targets in cancer. - Vu N Ngo;R Eric Davis;Laurence Lamy;Xin Yu;Hong Zhao;Georg Lenz;Lloyd T Lam;Sandeep Dave;Liming Yang;John Powell;Louis M Staudt - Nature (2006)
  9. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. - Martin Jinek;Krzysztof Chylinski;Ines Fonfara;Michael Hauer;Jennifer A Doudna;Emmanuelle Charpentier - Science (New York, N.Y.) (2012)
  10. Haploid genetic screens in human cells identify host factors used by pathogens. - Jan E Carette;Carla P Guimaraes;Malini Varadarajan;Annie S Park;Irene Wuethrich;Alzbeta Godarova;Maciej Kotecki;Brent H Cochran;Eric Spooner;Hidde L Ploegh;Thijn R Brummelkamp - Science (New York, N.Y.) (2009)

Literatures Citing This Work ​

  1. Progress in genomics according to bingo: 2013 edition. - Konrad J Karczewski - Genome biology (2013)
  2. Cas9-based tools for targeted genome editing and transcriptional control. - Tao Xu;Yongchao Li;Joy D Van Nostrand;Zhili He;Jizhong Zhou - Applied and environmental microbiology (2014)
  3. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. - Yanfang Fu;Jeffry D Sander;Deepak Reyon;Vincent M Cascio;J Keith Joung - Nature biotechnology (2014)
  4. Dissecting mammalian immunity through mutation. - Owen M Siggs - Immunology and cell biology (2014)
  5. Crystal structure of Cas9 in complex with guide RNA and target DNA. - Hiroshi Nishimasu;F Ann Ran;Patrick D Hsu;Silvana Konermann;Soraya I Shehata;Naoshi Dohmae;Ryuichiro Ishitani;Feng Zhang;Osamu Nureki - Cell (2014)
  6. Bacterial cellular engineering by genome editing and gene silencing. - Nobutaka Nakashima;Kentaro Miyazaki - International journal of molecular sciences (2014)
  7. CRISPR-based technologies: prokaryotic defense weapons repurposed. - Rebecca M Terns;Michael P Terns - Trends in genetics : TIG (2014)
  8. CRISPR-Cas systems for editing, regulating and targeting genomes. - Jeffry D Sander;J Keith Joung - Nature biotechnology (2014)
  9. When a virus is not a parasite: the beneficial effects of prophages on bacterial fitness. - Joseph Bondy-Denomy;Alan R Davidson - Journal of microbiology (Seoul, Korea) (2014)
  10. CRISPR-Cas system: a powerful tool for genome engineering. - Liang Liu;Xiu-Duo Fan - Plant molecular biology (2014)

... (1542 more literatures)

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