Genome Editing

  • Praveen P. Balgir
  • Suman Rani
  • Vishal


Understanding of natural DNA repair processes in cells has led to development of a variety of genome editing platforms. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are proving to be precise tools for genome engineering. These are basically chimeric nucleases, comprising interchangeable target-dependent, sequence-specific DNA-binding domains linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a number of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining (NHEJ) or homology-directed (HR) repair, at the specific targeted genomic loci. Clustered regularly interspaced short palindromic repeat (CRISPR) loci have a protective function in prokaryotes, against invasion by phage and plasmid DNA, acting through a genetic interference pathway. CRISPR loci have been located in 40% bacterial and 90% of archaeal genomes. These are observed to evolve and adapt rapidly by acquiring new spacer sequences on exposure to highly dynamic genomes of phages. This bacterial immune system matches the spacer sequences located between CRISPR repeat sequences and the invading DNA, to carry out its interference activity. Each CRISPR cluster is constituted of genetically linked subset of Cas (CRISPR-associated) genes, which collectively encode more than 40 families of proteins, involved in adaptation and interference. CRISPR loci are transcribed and converted into small CRISPR RNAs (crRNAs) that contain a full spacer sequence, flanked by partial repeat sequences of a complex of Cas proteins termed cascade (Cas-complex for antivirus defense). CrRNA spacers are known to bind target invading “protospacer” DNA by direct Watson–Crick base pairing leading to its degradation. The mechanism of CRISPR self-/non-self-discrimination involves target/crRNA mismatches at specific positions outside of the spacer sequence to identify foreign DNA for degradation, whereas extended pairing between crRNA and CRISPR DNA repeats is protective and prevents self-degradation. Various applications already built up on the platform technologies in different fields such as generation of animal models, treatment of infectious diseases, correction of genetic disorders, functional genome screening, and stem cell gene editing have been discussed.



This work was supported by grant from the DBT-Punjabi University Interdisciplinary Life Science Programme for advanced research and education (DBT-IPLS Project) No. BT/PR-4548/INF/22/146/2012.


  1. Bedell VM, Wang Y, Campbell JM, Poshusta TL et al (2012) In vivo genome editing using a high-efficiency TALEN system. Nature 491:114–118CrossRefPubMedPubMedCentralGoogle Scholar
  2. Beerli RR, Barbas CF (2002) Engineering polydactyl zinc finger transcription factors. Nat Biotechnol 20:135–141CrossRefPubMedGoogle Scholar
  3. Beerli RR, Dreier B, Barbas CF (2000) Positive and negative regulation of endogenous genes by designed transcription factors. Proc Natl Acad Sci USA 97(4):1495–1500CrossRefPubMedGoogle Scholar
  4. Bhaya D, Davison M, Barrangou R (2011) CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45:273–297CrossRefPubMedGoogle Scholar
  5. Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333(6051):1843–1846CrossRefPubMedGoogle Scholar
  6. Carlson DF, Tan W, Lillico SG, Stverakova D et al (2012) Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci USA 109:17382–17387CrossRefPubMedGoogle Scholar
  7. Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188:773–7823CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cermak T, Doyle EL, Christian M et al (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39(12):e82CrossRefPubMedPubMedCentralGoogle Scholar
  9. Denomy JB, Davidson AR (2014) To acquire or resist: the complex biological effects of CRISPR–Cas systems. Trends Microbiol 22(4):218–225CrossRefGoogle Scholar
  10. Ding Q, Lee YK, Schaefer EA, Peters DT et al (2013) A TALEN genome-editing system for generating human stem cell-based disease models. Stem Cells 12(2):225–238Google Scholar
  11. Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096CrossRefPubMedGoogle Scholar
  12. Gilbert LA, Horlbeck MA, Adamson B et al (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159(3):647–661CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hockemeyer D, Soldner F, Beard C et al (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27(9):851–857CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hockemeyer D, Wang H, Kiani S, Lai CS et al (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29(8):731–734CrossRefPubMedPubMedCentralGoogle Scholar
  15. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278CrossRefPubMedPubMedCentralGoogle Scholar
  16. Ishino Y, Shinagawa H, Makino K et al (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433CrossRefPubMedPubMedCentralGoogle Scholar
  17. Iwase H, Liu H, Wijkstrom M et al (2015) Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date. Xenotransplantation 22(4):302–309CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kim E, Kim S, Kim DH, Choi BS et al (2012) Precision genome engineering with programmable DNA-nicking enzymes. Genome Res 22(7):1327–1333CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kim Y, Kweon J, Kim A, Chon JK et al (2013) A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 31(3):251–258CrossRefPubMedGoogle Scholar
  20. Koonin EV, Wolf IY (2009) Is evolution Darwinian or/and Lamarckian? Biol Direct 4:42CrossRefPubMedPubMedCentralGoogle Scholar
  21. Li T, Huang S, Jiang WZ et al (2011) TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 39:359–372CrossRefPubMedGoogle Scholar
  22. Li T, Liu B, Spalding MH et al (2012) High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat Biotechnol 30:390–392CrossRefPubMedGoogle Scholar
  23. Liu Q, Segal DJ, Ghiara JB, Barbas CF (1997) Design of polydactyl zinc-finger proteins for unique addressing within complex genomes. Proc Natl Acad Sci USA 94(11):5525–5530CrossRefPubMedGoogle Scholar
  24. Liu J, Li C, Yu Z, Huang P et al (2012) Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. J Genet Genomics 39:209–215CrossRefPubMedGoogle Scholar
  25. Lombardo A, Genovese P, Beausejour CM et al (2007) Gene editing in human stem cells using zinc-finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol 25(11):1298–1306CrossRefGoogle Scholar
  26. Mahfouz MM, Li L, Shamimuzzaman M, Wibowo A et al (2011) De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc Natl Acad Sci USA 108:2623–2628CrossRefPubMedGoogle Scholar
  27. Makarova KS, Grishin NV, Shabalina SA et al (2006) A putative RNA interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1:7CrossRefPubMedPubMedCentralGoogle Scholar
  28. Makarova KS, Haft DH, Barrangou R et al (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9(6):467–477CrossRefPubMedGoogle Scholar
  29. Makarova KS, Wolf YI, Koonin EV (2013) The basic building blocks and evolution of CRISPR-cas systems. Biochem Soc T 41:1392–1400CrossRefGoogle Scholar
  30. Perez EE, Wang J, Miller JC et al (2008) Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 26(7):808–816CrossRefPubMedPubMedCentralGoogle Scholar
  31. Qi LS, Larson MH, Gilbert LA et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence specific control of gene expression. Cell 152(5):1173–1183CrossRefPubMedPubMedCentralGoogle Scholar
  32. Ramirez CL, Certo MT, Mussolino C, Goodwin MJ et al (2012) Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects. Nucleic Acids Res 40(12):5560–5568CrossRefPubMedPubMedCentralGoogle Scholar
  33. Richter H, Randau L, Plagens A (2013) Exploiting CRISPR/Cas: interference mechanisms and applications. Int J Mol Sci 14:14518–14531CrossRefPubMedPubMedCentralGoogle Scholar
  34. Scharenberg AM, Duchateau P, Smith J (2013) Genome engineering with TAL-effector nucleases and alternative modular nuclease technologies. Curr Gene Ther 13(4):291–303CrossRefPubMedGoogle Scholar
  35. Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem 82:237–266CrossRefPubMedGoogle Scholar
  36. Stoddard BL (2011) Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification. Structure 19(1):7–15CrossRefPubMedPubMedCentralGoogle Scholar
  37. Urnov FD, Rebar EJ, Holmes MC et al (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11(9):636–646CrossRefPubMedGoogle Scholar
  38. Wang J, Friedman G, Doyon Y et al (2012) Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme. Genome Res 22(7):1316–1326CrossRefPubMedPubMedCentralGoogle Scholar
  39. Wiedenheft B, Samuel H, Sternberg, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338CrossRefPubMedGoogle Scholar
  40. Wyman C, Kanaar R (2006) DNA double-strand break repair: all’s well that ends well. Annu Rev Genet 40:363–383CrossRefPubMedGoogle Scholar
  41. Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La Russa M et al (2015) Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160:339–350CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Praveen P. Balgir
    • 1
  • Suman Rani
    • 1
  • Vishal
    • 1
  1. 1.Department of BiotechnologyPunjabi UniversityPatialaIndia

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