Practical Considerations for Using Pooled Lentiviral CRISPR Libraries

Joel R. McDade1, Nicole C. Waxmonsky1, Lianna E. Swanson1, Melina Fan1

1 Addgene, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 31.5
DOI:  10.1002/cpmb.8
Online Posting Date:  July, 2016
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CRISPR/Cas9 technology is ideally suited for genome‐wide screening applications due to the ease of generating guide RNAs (gRNAs) and the versatility of Cas9 or Cas9 derivatives to knockout, repress, or activate expression of target genes. Several pooled lentiviral CRISPR libraries have been developed and are now publicly available, but while using CRISPR/Cas9 for genetic experiments has become widely adopted, genome‐wide screening experiments remain technically challenging. This review covers the basics of CRISPR/Cas9, describes several publicly available CRISPR libraries, and provides a general protocol for conducting genome‐wide screening experiments using CRISPR/Cas9. © 2016 by John Wiley & Sons, Inc.

Keywords: CRISPR/Cas9; genome‐wide screening; pooled libraries

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Table of Contents

  • The Basics of CRISPR/Cas9 In Genome Engineering
  • A Practical Guide for Pooled Lentiviral Crispr Libraries
  • Conclusions
  • Acknowledgements
  • Conflict Of Interest
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
  Anders, C., Niewoehner, O., Duerst, A., and Jinek, M. 2014. Structural basis of PAM‐dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569‐573. doi: 10.1038/nature13579.
  Bassett, A.R., Kong, L., and Liu, J.L. 2015. A genome‐wide CRISPR library for high‐throughput genetic screening in Drosophila cells. J. Genet. Genomics 42:301‐309. doi: 10.1016/j.jgg.2015.03.011.
  Boettcher, M. and McManus, M.T. 2015. Choosing the right tool for the job: RNAi, TALEN, or CRISPR. Cell 4:575‐585. doi: 10.1016/j.molcel.2015.04.028.
  Chavez, A., Scheiman, J., Vora, S., Pruitt, B.W., Tuttle, M., Iyer, E.P.R., Lin, S., Kiani, S., Guzman, C.D., Wiegand, D.J., Ter‐Ovanesyan, D., Braff, J.L., Davidsohn, N., Housden, B.E., Perrimon, N., Weiss, R., Aach, J., Collins, J.J., and Church, G.M. 2015. Highly efficient Cas9‐mediated transcriptional programming. Nat. Methods 12:326‐328. doi: 10.1038/nmeth.3312.
  Chen, S., Sanjana, N.E., Zheng, K., Shalem, O., Lee, K., Shi, X., Scott, D.A., Song, J., Pan, J.Q., Weissleder, R., Lee, H., Zhang, F., and Sharp, P.A. 2015. Genome‐wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160:1246‐1260. doi: 10.1016/j.cell.2015.02.038.
  Chu, V.T., Weber, T., Wefers, B., Wurst, W., Sander, S., Rajewsky, K., and Kühn, R. 2015. Increasing the efficiency of homology‐directed repair for CRISPR‐Cas9‐induced precise gene editing in mammalian cells. Nat. Biotechnol. 33:543‐548. doi: 10.1038/nbt.3198.
  Doench, J.G., Hartenian, E., Graham, D.B., Tothova, Z., Hegde, M., Smith, I., Sullender, M., Ebert, B.L., Xavier, R.J., and Root, D.E. 2014. Rational design of highly active sgRNAs for CRISPR‐Cas9‐mediated gene inactivation. Nat. Biotechnol. 32:1262‐1267. doi: 10.1038/nbt.3026.
  Doench, J.G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E.W., Donovan, K.F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., Virgin, H.W., Listgarten, J., and Root, D.E. 2016. Optimized sgRNA design to maximize activity and minimize off‐target effects of CRISPR‐Cas9. Nat. Biotechnol. 34:184‐191. doi: 10.1038/nbt.3437.
  Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L., Reyon, D., Joung, J.K., and Sander, J.D. 2013. High‐frequency off‐target mutagenesis induced by CRISPR‐Cas nucleases in human cells. Nat. Biotechnol. 31:822‐826. doi: 10.1038/nbt.2623.
  Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E., Stern‐Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J.A., Lim, W.A., Weissman, J.S., and Qi, L.S. 2013. CRISPR‐mediated modular RNA‐guided regulation of transcription in eukaryotes. Cell 154:442‐451. doi: 10.1016/j.cell.2013.06.044.
  Hart, T., Chandrashekhar, M., Aregger, M., Steinhart, Z., Brown, K.R., MacLeod, G., Mis, M., Zimmermann, M., Fradet‐Turcotte, A., Sun, S., Mero, P., Dirks, P., Sidhu, S., Roth, F.P., Rissland, O.S., Durocher, D., Angers, S., and Moffat, J. 2015. High‐resolution CRISPR screens reveal fitness genes and genotype‐specific cancer liabilities. Cell 163:1515‐1526. doi: 10.1016/j.cell.2015.11.015.
  Hsu, P.D., Scott, D.A., Weinstein, J.A., Ran, F.A., Konermann, S., Agarwala, V., Li, Y., Fine, E.J., Wu, X., Shalem, O., Cradick, T.J., Marraffini, L.A., Bao, G., and Zhang, F. 2013. DNA targeting specificity of RNA‐guided Cas9 nucleases. Nat. Biotechnol. 31:827‐832. doi: 10.1038/nbt.2647.
  Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., Peterson, R.T., Yeh, J.R., and Joung, J.K. 2013. Efficient genome editing in zebrafish using a CRISPR‐Cas system. Nat. Biotechnol. 31:227‐229. doi: 10.1038/nbt.2501.
  Kabadi, A.M., Ousterout, D.G., Hilton, I.B., and Gersbach, C.A. 2014. Multiplex CRISPR/Cas9‐based genome engineering from a single lentiviral vector. Nucleic Acids Res. 42:e147. doi: 10.1093/nar/gku749.
  Kleinstiver, B.P., Prew, M.S., Tsai, S.Q., Topkar, V.V., Nguyen, N.T., Zheng, Z., Gonzales, A.P., Li, Z., Peterson, R.T., Yeh, J.J., Aryee, M.J., and Joung, J.K. 2015. Engineered CRISPR‐Cas9 nucleases with altered PAM specificities. Nature 523:481‐485. doi: 10.1038/nature14592.
  Koike‐Yusa, H., Li, Y., Tan, E.P., Velasco‐Herrera, M., and Yusa, K. 2014. Genome‐wide recessive genetic screening in mammalian cells with a lentiviral CRISPR‐guide RNA library. Nat. Biotechnol. 32:267‐273. doi: 10.1038/nbt.2800.
  Konermann, S., Brigham, M.D., Trevino, A.E., Joung, J., Abudayyeh, O.O., Barcena, C., Hsu, P.D., Habib, N., Gootenberg, J.S., Nishimasu, H., Nureki, O., and Zhang, F. 2015. Genome‐scale transcriptional activation by an engineered CRISPR‐Cas9 complex. Nature 517:583‐588. doi: 10.1038/nature14136.
  Lin, S., Staahl, B.T., Alla, R.K., and Doudna, J.A. 2014. Enhanced homology‐directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3:e04766. doi: 10.7554/eLife.04766.
  Luo, B., Cheung, H.W., Subramanian, A., Sharifnia, T., Okamoto, M., Yang, X., Hinkle, G., Boehm, J.S., Beroukhim, R., Weir, B.A., Mermel, C., Barbie, D.A., Awad, T., Zhou, X., Nguyen, T., Piqani, B., Li, C., Golub, T.R., Meyerson, M., Hacohen, N., Hahn, W.C., Lander, E.S., Sabatini, D.M., and Root, D.E. 2008. Highly parallel identification of essential genes in cancer cells. Proc. Natl. Acad. Sci. U.S.A. 105:20380‐20385. doi: 10.1073/pnas.0810485105.
  Ma, H., Dang, Y., Wu, Y., Jia, G., Anaya, E., Zhang, J., Abraham, S., Choi, J.G., Shi, G., Qi, L., Manjunath, N., and Wu, H. 2015. A CRISPR‐Based Screen Identifies Genes essential for West‐Nile‐virus‐induced cell death. Cell Rep. 12:673‐683. doi: 10.1016/j.celrep.2015.06.049.
  Mali, P., Aach, J., Stranges, P.B., Esvelt, K.M., Moosburner, M., Kosuri, S., Yang, L., and Church, G.M. 2013a CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31:833‐838. doi: 10.1038/nbt.2675.
  Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., and Church, G.M. 2013b. RNA‐guided human genome engineering via Cas9. Science 339:823‐826. doi: 10.1126/science.1232033.
  Nishimasu, H., Ran, F.A., Hsu, P.D., Konermann, S., Shehata, S.I., Dohmae, N., Ishitani, R., Zhang, F., and Nureki, O. 2014. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156:935‐949. doi: 10.1016/j.cell.2014.02.001.
  Qi, L.S., Larson, M.H., Gilbert, L.A., Doudna, J.A., Weissman, J.S., Arkin, A.P., and Lim, W.A. 2013. Repurposing CRISPR as an RNA‐guided platform for sequence‐specific control of gene expression. Cell 152:1173‐1183. doi: 10.1016/j.cell.2013.02.022.
  Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., and Zhang, F. 2013. Genome engineering using the CRISPR‐Cas9 system. Nat. Protoc. 8:2281‐2308. doi: 10.1038/nprot.2013.143.
  Sanjana, N.E., Shalem, O., and Zhang, F. 2014. Improved vectors and genome‐wide libraries for CRISPR screening. Nat. Methods 8:783‐784. doi: 10.1038/nmeth.3047.
  Shalem, O., Sanjana, N.E., Hartenian, E., Shi, X., Scott, D.A., Mikkelsen, T.S., Heckl, D., Ebert, B.L., Root, D.E., Doench, J.G., and Zhang, F. 2014. Genome‐scale CRISPR‐Cas9 knockout screening in human cells. Science 343:84‐87. doi: 10.1126/science.1247005.
  Shalem, O., Sanjana, N.E., and Zhang, F. 2015. High‐throughput functional genomics using CRISPR‐Cas9. Nat. Rev. Genet. 16:299‐311. doi: 10.1038/nrg3899.
  Shechner, D.M., Hacisuleyman, E., Younger, S.T., and Rinn, J.L. 2015. Multiplexable, locus‐specific targeting of long RNAs with CRISPR‐Display. Nat. Methods 12:664‐670. doi: 10.1038/nmeth.3433.
  Shi, J., Wang, E., Milazzo, J.P., Wang, Z., Kinney, J.B., and Vakoc, C.R. 2015. Discovery of cancer drug targets by CRISPR‐Cas9 screening of protein domains. Nat. Biotechnol. 33:661‐667. doi: 10.1038/nbt.3235.
  Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S., and Mesirov, J.P. 2005. Gene set enrichment analysis: A knowledge‐based approach for interpreting genome‐wide expression profiles. Proc. Natl. Acad. Sci. U.S.A. 102:15545‐15550. doi: 10.1073/pnas.0506580102.
  Tanenbaum, M.E., Gilbert, L.A., Qi, L.S., Weissman, J.S., and Vale, R.D. 2014. A protein‐tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635‐646. doi: 10.1016/j.cell.2014.09.039.
  Wang, T., Wei, J.J., Sabatini, D.M., and Lander, E.S. 2014. Genetic screens in human cells using the CRISPR‐Cas9 system. Science 343:80‐84. doi: 10.1126/science.1246981.
  Wang, T., Birsoy, K., Hughes, N.W., Krupczak, K.M., Post, Y., Wei, J.J., Lander, E.S., and Sabatini, D.M. 2015. Identification and characterization of essential genes in the human genome. Science 350:1096‐1101. doi: 10.1126/science.aac7041.
  Zhong, C., Yin, Q., Xie, Z., Bai, M., Dong, R., Tang, W., Xing, Y.‐H., Zhang, H., Yang, S., Chen, L.‐L., Bartolomei, M.S., Ferguson‐Smith, A., Li, D., Yang, L., Wu, Y., and Li, J. 2015. CRISPR‐Cas9‐mediated genetic screening in mice with haploid embryonic stem cells carrying a guide RNA library. Cell Stem Cell 17:221‐232. doi: 10.1016/j.stem.2015.06.005.
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