Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

Abstract

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

Shown for reading. Its publisher allows quoting it, not republishing it.

References 25

1. 1.

   Aguirre A. J. Meyers R. M. Weir B. A. Vazquez F. Zhang C. Z., 2016 Genomic copy number dictates a gene-independent cell response to CRISPR-Cas9 targeting. Cancer Discov. 6: 914–929.

   Not in our library10.1158/2159-8290.cd-16-0154

2. 2.

   Blomen V. A. Majek P. Jae L. T. Bigenzahn J. W. Nieuwenhuis J., 2015 Gene essentiality and synthetic lethality in haploid human cells. Science 350: 1092–1096.

   Not in our library10.1126/science.aac7557

3. 3.

   Doench J. G. Fusi N. Sullender M. Hegde M. Vaimberg E. W., 2016 Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34: 184–191.

   Not in our library10.1038/nbt.3437

4. 4.

   Echeverri C. J. Beachy P. A. Baum B. Boutros M. Buchholz F., 2006 Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat. Methods 3: 777–779.

   Not in our library10.1038/nmeth1006-777

5. 5.

   Evers B. Jastrzebski K. Heijmans J. P. Grernrum W. Beijersbergen R. L., 2016 CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nat. Biotechnol. 34: 631–633.

   Not in our library10.1038/nbt.3536

6. 6.
   Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR · 2016

   No notice
7. 7.
   BAGEL: a computational framework for identifying essential genes from pooled library screens · 2016

   No notice
8. 8.
   Measuring error rates in genomic perturbation screens: gold standards for human functional genomics · 2014

   No notice
9. 9.

   Hart T. Chandrashekhar M. Aregger M. Steinhart Z. Brown K. R., 2015 High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163: 1515–1526.

   Not in our library10.1016/j.cell.2015.11.015

10. 10.

    Heigwer F. Kerr G. Boutros M., 2014 E-CRISP: fast CRISPR target site identification. Nat. Methods 11: 122–123.

    Not in our library10.1038/nmeth.2812

11. 11.

    Heigwer F. Zhan T. Breinig M. Winter J. Brugemann D., 2016 CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries. Genome Biol. 17: 55.

    Not in our library10.1186/s13059-016-0915-2

12. 12.

    Hsu P. D. Scott D. A. Weinstein J. A. Ran F. A. Konermann S., 2013 DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31: 827–832.

    Not in our library10.1038/nbt.2647

13. 13.

    Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J. A., 2012 A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816–821.

    Not in our library10.1126/science.1225829

14. 14.

    Kaelin W. G. Jr, 2012 Molecular biology. Use and abuse of RNAi to study mammalian gene function. Science 337: 421–422.

    Not in our library10.1126/science.1225787

15. 15.

    Koike-Yusa H. Li Y. Tan E. P. Velasco-Herrera Mdel C. Yusa K., 2014 Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32: 267–273.

    Not in our library10.1038/nbt.2800

16. 16.
    Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system · 2016

    No notice
17. 17.

    Munoz D. M. Cassiani P. J. Li L. Billy E. Korn J. M., 2016 CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions. Cancer Discov. 6: 900–913.

    Not in our library10.1158/2159-8290.cd-16-0178

18. 18.

    Park J. Kim J. S. Bae S., 2016 Cas-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cas9. Bioinformatics 32: 2017–2023.

    Not in our library10.1093/bioinformatics/btw103

19. 19.

    Parnas O. Jovanovic M. Eisenhaure T. M. Herbst R. H. Dixit A., 2015 A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162: 675–686.

    Not in our library10.1016/j.cell.2015.06.059

20. 20.

    Shalem O. Sanjana N. E. Hartenian E. Shi X. Scott D. A., 2014 Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343: 84–87.

    Not in our library10.1126/science.1247005

21. 21.

    Steinhart Z. Pavlovic Z. Chandrashekhar M. Hart T. Wang X., 2017 Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors. Nat. Med. 23: 60–68.

    Not in our library10.1038/nm.4219

22. 22.
    A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia · 2016

    No notice
23. 23.

    Wang T. Wei J. J. Sabatini D. M. Lander E. S., 2014 Genetic screens in human cells using the CRISPR-Cas9 system. Science 343: 80–84.

    Not in our library10.1126/science.1246981

24. 24.

    Wang T. Birsoy K. Hughes N. W. Krupczak K. M. Post Y., 2015 Identification and characterization of essential genes in the human genome. Science 350: 1096–1101.

    Not in our library10.1126/science.aac7041

25. 25.

    Wang T. Yu H. Hughes N. W. Liu B. Kendirli A., 2017 Gene essentiality profiling reveals gene networks and synthetic lethal interactions with Oncogenic Ras. Cell 168: 890–903 e15.

    Not in our library10.1016/j.cell.2017.01.013

Cited by papers we hold 17

• Accurate rare variant phasing of whole-genome and whole-exome sequencing data in the UK Biobank · 2023
  No notice
• Reduced gene templates for supervised analysis of scale-limited CRISPR-Cas9 fitness screens · 2022
  Not checked
• A comprehensive Bioconductor ecosystem for the design of CRISPR guide RNAs across nucleases and technologies · 2022
  Not checked
• CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition · 2022
  No notice
• Combinatorial CRISPR screen identifies fitness effects of gene paralogues · 2021
  No notice
• Discovery of putative tumor suppressors from CRISPR screens reveals rewired lipid metabolism in acute myeloid leukemia cells · 2021
  Not checked
• Improved analysis of CRISPR fitness screens and reduced off-target effects with the BAGEL2 gene essentiality classifier · 2021
  No notice
• Integrated cross-study datasets of genetic dependencies in cancer · 2021
  No notice
• Evaluating drug targets through human loss-of-function genetic variation · 2020
  Corrected
• The mutational constraint spectrum quantified from variation in 141,456 humans · 2020
  Corrected
• USP30 sets a trigger threshold for PINK1–PARKIN amplification of mitochondrial ubiquitylation · 2020
  No notice
• DEG 15, an update of the Database of Essential Genes that includes built-in analysis tools · 2020
  No notice
• Digital microfluidic isolation of single cells for -Omics · 2020
  No notice
• Copper bioavailability is a KRAS-specific vulnerability in colorectal cancer · 2020
  No notice
• The Network of Cancer Genes (NCG): a comprehensive catalogue of known and candidate cancer genes from cancer sequencing screens · 2019
  No notice
• Identifying chemogenetic interactions from CRISPR screens with drugZ · 2019
  No notice
• Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities · 2018
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.