Introduction to Gene Editing and Manipulation Using CRISPR/Cas9 Technology

Martin Newman1, Frederick M. Ausubel1

1 Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 31.4
DOI:  10.1002/cpmb.14
Online Posting Date:  July, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Until very recently, the prospect of introducing mutations or exogenous DNA sequences at precise locations in the genomes of plants and animals was difficult, if not impossible. This rapidly changed with the demonstration that the type II CRISPR‐Cas complex, a bacterial anti‐viral surveillance system, could be engineered into a simple and robust platform for introducing double‐stranded DNA breaks at nearly any position of plant and animal genomes. The prospect of efficiently creating tailored changes to a gene of interest is revolutionizing biomedical research, allowing exciting new questions to be asked. This overview introduces CRISPR‐Cas technology as a tool for molecular biology and briefly discusses the advantages of this method over earlier techniques, as well as unique opportunities to create new avenues of research. © 2016 by John Wiley & Sons, Inc.

Keywords: CRISPR; Cas9; sgRNA; genome editing

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Gene Editing and Manipulation Using Crispr/Cas9 Technology
  • Acknowledgements
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Bartlett, J.M.S. and Stirling, D. (eds.) 2003. A short history of the polymerase chain reaction. In Methods in Molecular Biology Vol. 226, PCR Protocols, 2nd edition. pp. 3‐6. Humana Press, Totowa, N.J.
  Bell, R.T., Fu, B.X., and Fire, A.Z. 2015. Cas9 variants expand the target repertoire in Caenorhabditis elegans. Genetics 202:381‐388. doi: 10.1534/genetics.115.185041.
  Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A., and Bonas, U. 2009. Breaking the code of DNA binding specificity of TAL‐type III effectors. Science 326:1509‐1512. doi: 10.1126/science.1178811.
  Carroll, D. 2011. Genome engineering with zinc‐finger nucleases. Genetics 188:773‐782. doi: 10.1534/genetics.111.131433.
  Chen, B., Gilbert, L.A., Cimini, B.A., Schnitzbauer, J., Zhang, W., Li, G.W., Park, J., Blackburn, E.H., Weissman, J.S., Qi, L.S., and Huang, B. 2013. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479‐1491. doi: 10.1016/j.cell.2013.12.001.
  Cho, S.W., Lee, J., Carroll, D., Kim, J.S., and Lee, J. 2013. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9‐sgRNA ribonucleoproteins. Genetics 195:1177‐1180. doi: 10.1534/genetics.113.155853.
  Cohen, S.N., Chang, A.C., Boyer, H.W., and Helling, R.B. 1973. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. U.S.A. 70:3240‐3244. doi: 10.1073/pnas.70.11.3240.
  Danna, K. and Nathans, D. 1971. Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. U.S.A. 68:2913‐2917. doi: 10.1073/pnas.68.12.2913.
  Dickinson, D.J., Ward, J.D., Reiner, D.J., and Goldstein, B. 2013. Engineering the Caenorhabditis elegans genome using Cas9‐triggered homologous recombination. Nat. Methods 10:1028‐1034. doi: 10.1038/nmeth.2641.
  Doudna, J.A. and Charpentier, E. 2014. Genome editing. The new frontier of genome engineering with CRISPR‐Cas9. Science 346:1258096. doi: 10.1126/science.1258096.
  Friedland, A.E., Tzur, Y.B., Esvelt, K.M., Colaiacovo, M.P., Church, G.M., and Calarco, J.A. 2013. Heritable genome editing in C. elegans via a CRISPR‐Cas9 system. Nat. Methods 10:741‐743. doi: 10.1038/nmeth.2532.
  Gantz, V.M. and Bier, E. 2015. Genome editing. The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science 348:442‐444. doi: 10.1126/science.aaa5945.
  Gratz, S.J., Rubinstein, C.D., Harrison, M.M., Wildonger, J., and O'Connor‐Giles, K.M. 2015. CRISPR‐Cas9 genome editing in Drosophila. Curr. Protoc. Mol. Biol. 111:31.2.1‐31.2.20. doi: 10.1002/0471142727.mb3102s111.
  Hsu, P.D., Lander, E.S., and Zhang, F. 2014. Development and applications of CRISPR‐Cas9 for genome engineering. Cell 157:1262‐1278. doi: 10.1016/j.cell.2014.05.010.
  Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. 2012. A programmable dual‐RNA‐guided DNA endonuclease in adaptive bacterial immunity. Science 337:816‐821. doi: 10.1126/science.1225829.
  Kim, H.‐M. and Colaiácovo, M.P. 2016. CRISPR‐Cas9‐guided genome engineering in C. elegans. Curr. Protoc. Mol. Biol. 115:31.7.1‐31.7.16. doi: 10.1002/cpmb.7.
  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.R., Aryee, M.J., and Joung, J.K. 2015. Engineered CRISPR‐Cas9 nucleases with altered PAM specificities. Nature 523:481‐485. doi: 10.1038/nature14592.
  Lander, E.S. 2016. The heroes of CRISPR. Cell 164:18‐28. doi: 10.1016/j.cell.2015.12.041.
  Lo, T.W., Pickle, C.S., Lin, S., Ralston, E.J., Gurling, M., Schartner, C.M., Bian, Q., Doudna, J.A., and Meyer, B.J. 2013. Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics 195:331‐348. doi: 10.1534/genetics.113.155382.
  Ma, X. and Liu, Y.‐G. 2016. CRISPR/Cas9‐based multiplex genome editing in monocot and dicot plants. Curr. Protoc. Mol. Biol. 115:31.6.1‐31.6.21. doi: 10.1002/cpmb.10.
  Marraffini, L.A. 2015. CRISPR‐Cas immunity in prokaryotes. Nature 526:55‐61. doi: 10.1038/nature15386.
  Maxam, A.M. and Gilbert, W. 1977. A new method for sequencing DNA. Proc. Natl. Acad. Sci. U.S.A. 74:560‐564. doi: 10.1073/pnas.74.2.560.
  McDade, J.R., Waxmonsky, N.C., Swanson, L., and Fan, M. 2016. Practical considerations for using pooled lentiviral CRISPR libraries. Curr. Protoc. Mol. Biol. 115:31.5.1‐31.5.14. doi: 10.1002/cpmb.8.
  Mojica, F.J., Ferrer, C., Juez, G., and Rodriguez‐Valera, F. 1995. Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning. Mol. Microbiol. 17:85‐93. doi: 10.1111/j.1365‐2958.1995.mmi_17010085.x.
  Paix, A., Wang, Y., Smith, H.E., Lee, C.Y., Calidas, D., Lu, T., Smith, J., Schmidt, H., Krause, M.W., and Seydoux, G. 2014. Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 sites in Caenorhabditis elegans. Genetics 198:1347‐1356. doi: 10.1534/genetics.114.170423.
  Reyon, D., Khayter, C., Regan, M.R., Joung, J.K., and Sander, J.D. 2012. Engineering designer transcription activator‐like effector nucleases (TALENs) by REAL or REAL‐Fast assembly. Curr. Protoc. Mol. Biol. 100:12.15.1‐12.15.14. doi: 10.1002/0471142727.mb1215s100.
  Sander, J.D. and Joung, J.K. 2014. CRISPR‐Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32:347‐355. doi: 10.1038/nbt.2842.
  Sander, J.D., Maeder, M.L., and Joung, J.K. 2011. Engineering designer nucleases with customized cleavage specificities. Curr. Protoc. Mol. Biol. 96:12.13.1‐12.13.16. doi: 10.1002/0471142727.mb1213s96.
  Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain‐terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74:5463‐5467. doi: 10.1073/pnas.74.12.5463.
  Selle, K. and Barrangou, R. 2015. Harnessing CRISPR‐Cas systems for bacterial genome editing. Trends Microbiol. 23:225‐232. doi: 10.1016/j.tim.2015.01.008.
  Smith, H.O. and Wilcox, K.W. 1970. A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J. Mol. Biol. 51:379‐391. doi: 10.1016/0022‐2836(70)90149‐X.
  Wyvekens, N., Tsai, S.Q., and Joung, J.K. 2015. Genome editing in human cells using CRISPR/Cas nucleases. Curr. Protoc. Mol. Biol. 112:31.3.1‐31.3.18. doi: 10.1002/0471142727.mb3103s112.
  Yang, L., Yang, J.L., Byrne, S., Pan, J., and Church, G.M. 2014. CRISPR/Cas9‐directed genome editing of cultured cells. Curr. Protoc. Mol. Biol. 107:31.1.1‐31.1.17. doi: 10.1002/0471142727.mb3101s107.
  Zhao, P., Zhang, Z., Ke, H., Yue, Y., and Xue, D. 2014. Oligonucleotide‐based targeted gene editing in C. elegans via the CRISPR/Cas9 system. Cell Res. 24:247‐250. doi: 10.1038/cr.2014.9.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library