CRISPR/Cas9 library screening for drug target discovery

Abstract

CRISPR/Cas9-based tools have rapidly developed in recent years. These include CRISPR-based gene activation (CRISPRa) or inhibition (CRISPRi), for which there are libraries. CRISPR libraries for loss of function have been widely used to identify new biological mechanisms, such as drug resistance and cell survival signals. CRISPRa is highly useful in screening for gain of functions, and CRISPRi is a more powerful tool than RNA interference (RNAi) libraries in screening for loss of functions. Positive selection using a CRISPR library can detect survival cells with specific conditions, such as drug treatment, and it can easily clarify drug resistance mechanisms. Negative selection is capable of detecting dead or slow-growing cells efficiently, and it can identify survival-essential genes, which can be promising candidates for molecularly targeted drugs. In addition, negative selection can be applied for synthetic lethality interactions, where the perturbation of both genes simultaneously results in the loss of viability, but that of either gene alone does not affect viability. This mechanism is highly important to identifying the optimal combination of molecularly targeted drugs. Survival-co-essential genes in cancer cells can be identified using new methods, such as the paired guide RNA system and in combination with single-cell RNA sequencing techniques. These efficient methods can clarify interesting biological mechanisms and suggest candidates for molecularly targeted drugs. This review identifies what types of screenings were performed and suggests ideas for the next CRISPR screenings to develop new drugs.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1

References

  1. 1.

    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR. Science. 2013;339:819–23.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013;23:1163–71.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013;152:1173–83.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154:442–51.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Lin S, Ewen-Campen B, Ni X, Housden BE, Perrimon N. In vivo transcriptional activation using CRISPR/Cas9 in Drosophila. Genetics. 2015;201:433–42.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2015;517:583–8.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014;159:635–46.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Hilton IB, D’Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol. 2015;33:510–7.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Chavez A, Tuttle M, Pruitt BW, Ewen-Campen B, Chari R, Ter-Ovanesyan D, et al. Comparison of Cas9 activators in multiple species. Nat Methods. 2016;13:563–7.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Nakamura T. Retroviral insertional mutagenesis identifies oncogene cooperation. Cancer Sci. 2005;96:7–12.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Largaespada DA. Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification. Methods. 2009;49:282–6.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Westbrook TF, Martin ES, Schlabach MR, Leng Y, Liang AC, Feng B, et al. A genetic screen for candidate tumor suppressors identifies REST. Cell. 2005;121:837–48.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Kolfschoten IG, van Leeuwen B, Berns K, Mullenders J, Beijersbergen RL, Bernards R, et al. A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell. 2005;121:849–58.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Ngo VN, Davis RE, Lamy L, Yu X, Zhao H, Lenz G, et al. A loss-of-function RNA interference screen for molecular targets in cancer. Nature. 2006;441:106–10.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Schlabach MR, Luo J, Solimini NL, Hu G, Xu Q, Li MZ, et al. Cancer proliferation gene discovery through functional genomics. Science. 2008;319:620–4.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Ranzani M, Annunziato S, Calabria A, Brasca S, Benedicenti F, Gallina P, et al. Lentiviral vector-based insertional mutagenesis identifies genes involved in the resistance to targeted anticancer therapies. Mol. Ther. 2014;22:2056–68.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Chen L, Stuart L, Ohsumi TK, Burgess S, Varshney GK, Dastur A, et al. Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics. BMC Cancer. 2013;13:93

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Khorashad JS, Eiring AM, Mason CC, Gantz KC, Bowler AD, Redwine HM, et al. shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance. Blood. 2015;125:1772–81.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343:84–7.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014;343:80–4.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Kurata M, Rathe SK, Bailey NJ, Aumann NK, Jones JM, Veldhuijzen GW, et al. Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML. Sci Rep. 2016;6:36199.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell. 2014;159:647–61.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Hart T, Chandrashekhar M, Aregger M, Steinhart Z, Brown KR, MacLeod G, et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell. 2015;163:1515–26.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Tzelepis K, KoikeYusa H, De Braekeleer E, Li Y, Metzakopian E, Dovey OM, et al. A CRISPR dropout screen identifies genetic vulnerabilities and therapeutic targets in acute myeloid leukemia. Cell Rep. 2016;17:1193–205.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Wang T, Birsoy K, Hughes NW, Krupczak KM, Post Y, Wei JJ, et al. Identification and characterization of essential genes in the human genome. Science. 2015;350:1096–101.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Ledford H. Cancer: rhe Ras renaissance. Nature. 2015;520:278–80.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Wang T, Yu H, Hughes NW, Liu B, Kendirli A, Klein K, et al. Gene essentiality profiling reveals gene networks and synthetic lethal interactions with oncogenic Ras. Cell. 2017;168:890–903.e15

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Horlbeck MA, Gilbert LA, Villalta JE, Adamson B, Pak RA, Chen Y, et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. Elife. 2016:5. https://doi.org/10.7554/eLife.19760.

  30. 30.

    Liu SJ, Horlbeck MA, Cho SW, Birk HS, Malatesta M, He D, et al. CRISPRi-based genome-scale identification of functional long non-coding RNA loci in human cells. Science. 355. https://doi.org/10.1126/science.aah7111.

  31. 31.

    Wong AS, Choi GC, Cui CH, Pregernig G, Milani P, Adam M, et al. Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM. Proc Natl Acad Sci USA. 2016;113:2544–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Han K, Jeng EE, Hess GT, Morgens DW, Li A, Bassik MC. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions. Nat Biotechnol. 2017;35:463–74.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Zhu S, Li W, Liu J, Chen CH, Liao Q, Xu P, et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library. Nat Biotechnol. 2016;34:1279–86.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Junker JP, van Oudenaarden A. Every cell is special: genome-wide studies add a new dimension to single-cell biology. Cell. 2014;157:8–11.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Datlinger P, Rendeiro AF, Schmidl C, Krausgruber T, Traxler P, Klughammer J, et al. Pooled CRISPR screening with single-cell transcriptome readout. Nat Methods. 2017;14:297–301.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36

    Dixit A, Parnas O, Li B, Chen J, Fulco CP, Jerby-Arnon L, et al. Perturb-Seq: dissecting molecular circuits with scalable single-cell rna profiling of pooled genetic screens. Cell. 2016;167:1853–66.e17.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Jaitin DA, Weiner A, Yofe I, Lara-Astiaso D, Keren-Shaul H, David E, et al. Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-Seq. Cell. 2016;167:1883–96.e15.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11:783–84.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Jain IH, Zazzeron L, Goli R, Alexa K, Schatzman Bone S, Dhillon H, et al. Hypoxia as a therapy for mitochondrial disease. Science. 2016;352:54–61.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Marceau CD, Puschnik AS, Majzoub K, Ooi YS, Brewer SM, Fuchs G, et al. Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens. Nature. 2016;535:159–63.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34:184–91.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Blondel CJ, Park JS, Hubbard TP, Pacheco AR, Kuehl CJ, Walsh MJ, et al. CRISPR/Cas9 screens reveal requirements for host cell sulfation and fucosylation in bacterial type iii secretion system-mediated cytotoxicity. Cell Host Microbe. 2016;20:226–37.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Zhou Y, Zhu S, Cai C, Yuan P, Li C, Huang Y, et al. High-throughput screening of a CRISPR. Nature. 2014;509:487–91.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Ma H, Dang Y, Wu Y, Jia G, Anaya E, Zhang J, et al. A CRISPR-based screen identifies genes essential for West Nile virus-induced cell death. Cell Rep. 2015;12:673–83.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Kim HS, Lee K, Bae S, Park J, Lee CK, Kim M, et al. CRISPR/Cas9-mediated gene-knockout screens and target identification via whole genome sequencing uncover host genes required for picornavirus infection. J Biol Chem. 2017;292:10664-71.

  46. 46.

    Park RJ, Wang T, Koundakjian D, Hultquist JF, Lamothe Molina P, Monel B, et al. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat Genet. 2017;49:193–203.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera Mdel C, Yusa K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol. 2014;32:267–73.

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Haga K, Fujimoto A, Takai-Todaka R, Miki M, Doan YH, Murakami K, et al. Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells. Proc Natl Acad Sci USA. 2016;113:E6248–55.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell. 2015;160:1246–60.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Morito Kurata.

Ethics declarations

Conflict of interest

D.A.L. is co-owner and advisor to NeoClone Biotechnology, Inc., B-MoGen Biotechnologies Inc., and Discovery Genomics, Inc. No resources or personnel from any company were involved in this research in any way. The remaining authors declare that they have no competing interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kurata, M., Yamamoto, K., Moriarity, B.S. et al. CRISPR/Cas9 library screening for drug target discovery. J Hum Genet 63, 179–186 (2018). https://doi.org/10.1038/s10038-017-0376-9

Download citation

Further reading

Search