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Human Genetics

, Volume 135, Issue 9, pp 971–976 | Cite as

CRISPR: a versatile tool for both forward and reverse genetics research

  • Channabasavaiah B. Gurumurthy
  • M’hamed Grati
  • Masato Ohtsuka
  • Samantha L. P. Schilit
  • Rolen M. Quadros
  • Xue Zhong Liu
Perspective
Part of the following topical collections:
  1. Genome Editing

Abstract

Human genetics research employs the two opposing approaches of forward and reverse genetics. While forward genetics identifies and links a mutation to an observed disease etiology, reverse genetics induces mutations in model organisms to study their role in disease. In most cases, causality for mutations identified by forward genetics is confirmed by reverse genetics through the development of genetically engineered animal models and an assessment of whether the model can recapitulate the disease. While many technological advances have helped improve these approaches, some gaps still remain. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated), which has emerged as a revolutionary genetic engineering tool, holds great promise for closing such gaps. By combining the benefits of forward and reverse genetics, it has dramatically expedited human genetics research. We provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples.

Keywords

Reverse Genetic Double Strand Break Reverse Genetic Approach Forward Genetic CRISPR System 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We thank Sidi Chen for his helpful comments on the manuscript. CBG’s lab is partially supported by National Institute of General Medical Sciences of the National Institutes of Health under Grant No. P20GM103471 and the Center for Humanized Mice from ORIP/DPCPSI/NIH/1R24OD018546-01. XZL’s lab is supported by R01 DC05575, R01 DC01246, and R01 DC012115 from the National Institutes of Health/National Institute on Deafness and Other Communication Disorders. SLPS is supported by the NSF Graduate Research Fellowship DGE1144152. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. We apologize to colleagues whose studies could not be cited because of space constraints.

References

  1. Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, Tanaka KF, Sakuma T, Yamamoto T, Tanaka K (2015) Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol. doi: 10.1186/s13059-015-0653-x PubMedPubMedCentralGoogle Scholar
  2. Angeli S, Lin X, Liu XZ (2012) Genetics of hearing and deafness. Anat Rec Adv Integr Anat Evol Biol 295:1812–1829. doi: 10.1002/ar.22579 CrossRefGoogle Scholar
  3. Bedell MA, Jenkins NA, Copeland NG (1997) Mouse models of human disease. Part I: techniques and resources for genetic analysis in mice. Genes Dev 11:1–10CrossRefPubMedGoogle Scholar
  4. Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song J, Pan JQ, Weissleder R, Lee H, Zhang F, Sharp PA (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 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chen Z-Y, Tao Y, Gao X, Hu Y, Dai P, Kong W, Liu D (2016) Restoration of hearing by CRISPR/Cas9-mediated genome editing in the Pmca2 deafness mouse model by protein delivery. Assoc Res Otolaryngol Abs 39:138–139Google Scholar
  6. Chiou S-H, Winters IP, Wang J, Naranjo S, Dudgeon C, Tamburini FB, Brady JJ, Yang D, Grüner BM, Chuang C-H, Caswell DR, Zeng H, Chu P, Kim GE, Carpizo DR, Kim SK, Winslow MM (2015) Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev 29:1576–1585. doi: 10.1101/gad.264861.115 CrossRefPubMedCentralGoogle Scholar
  7. Claussnitzer M, Dankel SN, Kim K-H, Quon G, Meuleman W, Haugen C, Glunk V, Sousa IS, Beaudry JL, Puviindran V, Abdennur NA, Liu J, Svensson P-A, Hsu Y-H, Drucker DJ, Mellgren G, Hui C-C, Hauner H, Kellis M (2015) FTO obesity variant circuitry and adipocyte browning in humans. N Engl J Med 373:895–907. doi: 10.1056/NEJMoa1502214 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Dow LE, Fisher J, O’Rourke KP, Muley A, Kastenhuber ER, Livshits G, Tschaharganeh DF, Socci ND, Lowe SW (2015) Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol 33:390–394. doi: 10.1038/nbt.3155 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Fujii W, Kawasaki K, Sugiura K, Naito K (2013) Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res 41:e187–e187. doi: 10.1093/nar/gkt772 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Grati M, Yan D, Raval MH, Walsh T, Ma Q, Chakchouk I, Kannan-Sundhari A, Mittal R, Masmoudi S, Blanton SH, Tekin M, King M-C, Yengo CM, Liu XZ (2016) Myo3a causes human dominant deafness and interacts with protocadherin 15-Cd2 isoform. Hum Mutat. doi: 10.1002/humu.22961 PubMedGoogle Scholar
  11. Gurumurthy CB, Quadros R, Sato M, Mashimo T, Lloyd KCK, Ohtsuka M (2016) CRISPR/Cas9 and the paradigm shift in mouse genome manipulation technologies. In: Turksen K (ed) Genome editing. doi: 10.1007/978-3-319-34148-4
  12. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70CrossRefPubMedGoogle Scholar
  13. Harms DW, Quadros RM, Seruggia D, Ohtsuka M, Takahashi G, Montoliu L, Gurumurthy CB (2014) Mouse genome editing using the CRISPR/Cas system. Curr Protoc Hum Genet Editor Board Jonathan Haines Al 83:15.7.1–15.7.27. doi: 10.1002/0471142905.hg1507s83 CrossRefGoogle Scholar
  14. Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL (2014) Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat Biotechnol 32:941–946. doi: 10.1038/nbt.2951 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jiang J, Zhang L, Zhou X, Chen X, Huang G, Li F, Wang R, Wu N, Yan Y, Tong C, Srivastava S, Wang Y, Liu H, Ying Q-L (2016) Induction of site-specific chromosomal translocations in embryonic stem cells by CRISPR/Cas9. Sci Rep 6:21918. doi: 10.1038/srep21918 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Kim H, Kim J-S (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15:321–334. doi: 10.1038/nrg3686 CrossRefPubMedGoogle Scholar
  17. Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, Hwang J, Kim J-I, Kim J-S (2015) Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods 12:237–243. doi: 10.1038/nmeth.3284 CrossRefPubMedGoogle Scholar
  18. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK (2016) High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490–495. doi: 10.1038/nature16526 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lin S, Staahl BT, Alla RK, Doudna JA (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. doi: 10.7554/eLife.04766
  20. Liu XZ, Yan D (2007) Ageing and hearing loss. J Pathol 211:188–197. doi: 10.1002/path.2102 CrossRefPubMedGoogle Scholar
  21. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J (2000) Mutations: types and causes. In: Molecular cell biology, 4th edn. W H Freeman, New YorkGoogle Scholar
  22. Maddalo D, Manchado E, Concepcion CP, Bonetti C, Vidigal JA, Han Y-C, Ogrodowski P, Crippa A, Rekhtman N, de Stanchina E, Lowe SW, Ventura A (2014) In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516:423–427. doi: 10.1038/nature13902 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL (2015) Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol 33:538–542. doi: 10.1038/nbt.3190 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Mianné J, Chessum L, Kumar S, Aguilar C, Codner G, Hutchison M, Parker A, Mallon A-M, Wells S, Simon MM, Teboul L, Brown SDM, Bowl MR (2016) Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair. Genome Med. doi: 10.1186/s13073-016-0273-4 PubMedPubMedCentralGoogle Scholar
  25. Miura H, Gurumurthy CB, Sato T, Sato M, Ohtsuka M (2015) CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA. Sci Rep 5:12799. doi: 10.1038/srep12799 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Moresco EMY, Li X, Beutler B (2013) Going forward with genetics. Am J Pathol 182:1462–1473. doi: 10.1016/j.ajpath.2013.02.002 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Morton CC, Nance WE (2006) Newborn hearing screening—a silent revolution. N Engl J Med 354:2151–2164. doi: 10.1056/NEJMra050700 CrossRefPubMedGoogle Scholar
  28. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Rivera RMC, Madhavan S, Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA (2016) In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351:403–407. doi: 10.1126/science.aad5143 CrossRefPubMedGoogle Scholar
  29. Orack JC, Deleidi M, Pitt D, Mahajan K, Nicholas JA, Boster AL, Racke MK, Comabella M, Watanabe F, Imitola J (2015) Concise review: modeling multiple sclerosis with stem cell biological platforms: toward functional validation of cellular and molecular phenotypes in inflammation-induced neurodegeneration. Stem Cells Transl Med 4:252–260. doi: 10.5966/sctm.2014-0133 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F (2014) CRISPR-Cas9 Knockin mice for genome editing and cancer modeling. Cell 159:440–455. doi: 10.1016/j.cell.2014.09.014 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Sánchez-Rivera FJ, Jacks T (2015) Applications of the CRISPR–Cas9 system in cancer biology. Nat Rev Cancer 15:387–395. doi: 10.1038/nrc3950 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Savić N, Schwank G (2016) Advances in therapeutic CRISPR/Cas9 genome editing. Transl Res 168:15–21. doi: 10.1016/j.trsl.2015.09.008 CrossRefGoogle Scholar
  33. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–87. doi: 10.1126/science.1247005 CrossRefPubMedGoogle Scholar
  34. Shalem O, Sanjana NE, Zhang F (2015) High-throughput functional genomics using CRISPR–Cas9. Nat Rev Genet 16:299–311. doi: 10.1038/nrg3899 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Shen B, Zhang J, Wu H, Wang J, Ma K., Li Z, Zhang X, Zhang P, Huang X (2013) Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 23:720–723. doi: 10.1038/cr.2013.46 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS, Semenova E, Minakhin L, Joung J, Konermann S, Severinov K, Zhang F, Koonin EV (2015) Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol Cell 60:385–397. doi: 10.1016/j.molcel.2015.10.008 CrossRefPubMedGoogle Scholar
  37. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F (2016) Rationally engineered Cas9 nucleases with improved specificity. Science 351:84–88. doi: 10.1126/science.aad5227 CrossRefPubMedGoogle Scholar
  38. Spiden SL, Bortolozzi M, Di Leva F, de Angelis MH, Fuchs H, Lim D, Ortolano S, Ingham NJ, Brini M, Carafoli E, Mammano F, Steel KP (2008) The novel mouse mutation Oblivion inactivates the PMCA2 pump and causes progressive hearing loss. PLoS Genet 4:e1000238. doi: 10.1371/journal.pgen.1000238 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, Wyvekens N, Khayter C, Iafrate AJ, Le LP, Aryee MJ, Joung JK (2014) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33:187–197. doi: 10.1038/nbt.3117 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918. doi: 10.1016/j.cell.2013.04.025 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84. doi: 10.1126/science.1246981 CrossRefPubMedGoogle Scholar
  42. Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, Cai W, Yang G, Bronson R, Crowley DG, Zhang F, Anderson DG, Sharp PA, Jacks T (2014) CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514:380–384. doi: 10.1038/nature13589 CrossRefPubMedCentralGoogle Scholar
  43. Yan D, Liu X-Z (2010) Modifiers of hearing impairment in humans and mice. Curr Genom 11:269–278. doi: 10.2174/138920210791233054 CrossRefGoogle Scholar
  44. Yan J, Enge M, Whitington T, Dave K, Liu J, Sur I, Schmierer B, Jolma A, Kivioja T, Taipale M, Taipale J (2013) Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites. Cell 154:801–813. doi: 10.1016/j.cell.2013.07.034 CrossRefPubMedGoogle Scholar
  45. Yang Y, Wang L, Bell P, McMenamin D, He Z, White J, Yu H, Xu C, Morizono H, Musunuru K, Batshaw ML, Wilson JM (2016) A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol 34:334–338. doi: 10.1038/nbt.3469 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Yin H, Song C-Q, Dorkin JR, Zhu LJ, Li Y, Wu Q, Park A, Yang J, Suresh S, Bizhanova A, Gupta A, Bolukbasi MF, Walsh S, Bogorad RL, Gao G, Weng Z, Dong Y, Koteliansky V, Wolfe SA, Langer R, Xue W, Anderson DG (2016) Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol 34:328–333. doi: 10.1038/nbt.3471 CrossRefPubMedGoogle Scholar
  47. Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T (2016) ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Commun 7:10431. doi: 10.1038/ncomms10431 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 Is a single rna-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771. doi: 10.1016/j.cell.2015.09.038 CrossRefPubMedGoogle Scholar
  49. Zhang L, Jia R, Palange NJ, Satheka AC, Togo J, An Y, Humphrey M, Ban L, Ji Y, Jin H, Feng X, Zheng Y (2015) Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9. PLoS One 10:e0120396. doi: 10.1371/journal.pone.0120396 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Zhou Y, Zhu S, Cai C, Yuan P, Li C, Huang Y, Wei W (2014) High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509:487–491. doi: 10.1038/nature13166 CrossRefPubMedGoogle Scholar
  51. Zou B, Mittal R, Grati M, Lu Z, Shu Y, Tao Y, Feng Y, Xie D, Kong W, Yang S, Chen Z-Y, Liu X (2015) The application of genome editing in studying hearing loss. Hear Res 327:102–108. doi: 10.1016/j.heares.2015.04.016 CrossRefPubMedGoogle Scholar
  52. Zuckermann M, Hovestadt V, Knobbe-Thomsen CB, Zapatka M, Northcott PA, Schramm K, Belic J, Jones DTW, Tschida B, Moriarity B, Largaespada D, Roussel MF, Korshunov A, Reifenberger G, Pfister SM, Lichter P, Kawauchi D, Gronych J (2015) Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling. Nat Commun 6:7391. doi: 10.1038/ncomms8391 CrossRefPubMedPubMedCentralGoogle Scholar
  53. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen Z-Y, Liu DR (2014) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33:73–80. doi: 10.1038/nbt.3081 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Channabasavaiah B. Gurumurthy
    • 1
    • 2
  • M’hamed Grati
    • 3
  • Masato Ohtsuka
    • 4
    • 5
  • Samantha L. P. Schilit
    • 6
  • Rolen M. Quadros
    • 2
  • Xue Zhong Liu
    • 3
    • 7
  1. 1.Developmental NeuroscienceMunroe Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical CenterOmahaUSA
  2. 2.Mouse Genome Engineering Core Facility, Vice Chancellor for Research OfficeUniversity of Nebraska Medical CenterOmahaUSA
  3. 3.Otolaryngology DepartmentUniversity of Miami Miller School of MedicineMiamiUSA
  4. 4.Department of Molecular Life Science, Division of Basic Medical Science and Molecular MedicineSchool of Medicine, Tokai UniversityIsehara, KanagawaJapan
  5. 5.The Institute of Medical SciencesTokai UniversityIsehara, KanagawaJapan
  6. 6.Department of GeneticsHarvard Medical SchoolBostonUSA
  7. 7.Department of OtolaryngologyXiangya Hospital, Central South UniversityChangshaChina

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