Current Genetics

, Volume 64, Issue 6, pp 1229–1238 | Cite as

Scarless genome editing: progress towards understanding genotype–phenotype relationships

  • Gregory L. Elison
  • Murat AcarEmail author


The ability to predict phenotype from genotype has been an elusive goal for the biological sciences for several decades. Progress decoding genotype–phenotype relationships has been hampered by the challenge of introducing precise genetic changes to specific genomic locations. Here we provide a comparative review of the major techniques that have been historically used to make genetic changes in cells as well as the development of the CRISPR technology which enabled the ability to make marker-free disruptions in endogenous genomic locations. We also discuss how the achievement of truly scarless genome editing has required further adjustments of the original CRISPR method. We conclude by examining recently developed genome editing methods which are not reliant on the induction of a DNA double strand break and discuss the future of both genome engineering and the study of genotype–phenotype relationships.


Genome editing Genotype-Phenotype relationships CRISPR 



GLE was in part supported by the National Institute of Health T32 GM007499.


Directed Evolution

A process in which a specific DNA element (often an open reading frame or enzymatic binding site on a protein) is mutagenized to create a large library. This library is then put through a specific in vitro screening process, generally for multiple rounds, to identify mutations which give rise to a desired effect on the assayed activity or function

DNA Binding Motif

A region of a protein which is capable of binding to a specific DNA sequence

Donor Template

An (often) short piece of DNA containing desired DNA edits flanked by regions homologous to the region to be edited. Cells may use it as a repair template during homologous -recombination based DNA-repair mechanism

Double Strand Break

A type of DNA damage in which both strands have been severed in close proximity to each other. This results in the creation of two distinct strands from the original

Endogenous DNA

The native DNA of a cell

Genome Editing

The process of intentionally altering (adding, removing, or changing) the genome of a cell

Genome Scarring

The introduction of unwanted or unintended edits to cellular DNA during genome editing


The DNA composition of a genomic region or of the entire genome

Homologous recombination based DNA repair

A type of DNA damage repair mechanism in which the cell uses a homologous region to the one which has been cut as a template to repair the cut strand. During natural repair, this template is generally the sister chromosome of the one being cut, but during genome editing this template is generally synthetic DNA introduced to the cell for this purpose


The process of inducing random mutations to a piece of DNA or to an entire genome. Historically this has been done using radiation or mutagenic chemicals. A mutagenized stretch of DNA will contain a number of random mutations of all types


A protein which is capable of inducing a DNA double strand break


A behavior of a cell which is observable to a researcher

Plasmid-based expression system

A means of gene expression in which the gene of interest is placed into a non-integrating plasmid and transformed into a cell


  1. Andrianantoandro E, Basu S, Karig DK, Weiss R (2006) Synthetic biology: new engineering rules for an emerging discipline. Mol Syst Biol 2:0028, (2006)CrossRefPubMedGoogle Scholar
  2. Arnold FH (1993) Engineering proteins for nonnatural environments. FASEB J 7:744–749CrossRefGoogle Scholar
  3. Barbieri EM, Muir P, Akhuetie-Oni BO, Yellman CM, Isaacs FJ (2017) Precise editing at DNA replication forks enables multiplex genome engineering in Eukaryotes. Cell 171(e1413):1453–1467. CrossRefPubMedGoogle Scholar
  4. Barrangou R et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712. CrossRefGoogle Scholar
  5. Benoist C, Chambon P (1981) In vivo sequence requirements of the SV40 early promotor region. Nature 290:304–310CrossRefGoogle Scholar
  6. Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764. CrossRefPubMedGoogle Scholar
  7. Boeke JD, Trueheart J, Natsoulis G, Fink GR (1987) 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol 154:164–175CrossRefGoogle Scholar
  8. BRAM RJ, KORNBERG RD (1985) Specific protein binding to far upstream activating sequences in polymerase II promoters. Proc Natl Acad Sci USA 82:43–47CrossRefGoogle Scholar
  9. Brouns SJ et al (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Bush WS, Oetjens MT, Crawford DC (2016) Unravelling the human genome-phenome relationship using phenome-wide association studies. Nat Rev Genet 17:129–145. CrossRefPubMedGoogle Scholar
  11. Carey LB, van Dijk D, Sloot PM, Kaandorp JA, Segal E (2013) Promoter sequence determines the relationship between expression level and noise. PLoS Biol 11:e1001528. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Christian, M et al (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Collinge DB, Lund OS, Thordal-Christensen H (2008) What are the prospects for genetically engineered, disease resistant plants? Eur J Plant Pathol 121:217–231. CrossRefGoogle Scholar
  14. DiCarlo JE et al (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41:4336–4343. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Dill KA, MacCallum JL (2012) The protein-folding problem, 50 years on. Science 338:1042–1046CrossRefGoogle Scholar
  16. DOUGLAS HC, CONDIE F (1954) The genetic control of galactose utilization in Saccharomyces. J Bacteriol 68:662–670PubMedPubMedCentralGoogle Scholar
  17. Elison GL, Song R, Acar MA (2017) Precise genome editing method reveals insights into the activity of eukaryotic promoters. Cell Rep 18:275–286. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Elledge SJ, Davis RW (1988) A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene 70:303–312CrossRefGoogle Scholar
  19. Fraser PD, Enfissi EM, Bramley PM (2009) Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches. Arch Biochem Biophys 483:196–204. CrossRefPubMedGoogle Scholar
  20. Fridman Y et al (2010) Subtle alterations in PCNA-partner interactions severely impair DNA replication and repair. PLoS Biol 8:e1000507. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Friedmann T, Roblin R (1972) Gene therapy for human genetic disease? Science 175:949–955CrossRefGoogle Scholar
  22. Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 109:E2579-2586. CrossRefGoogle Scholar
  23. Hirschhorn JN, Lohmueller K, Byrne E, Hirschhorn K (2002) A comprehensive review of genetic association studies. Genet Med 4:45–61CrossRefGoogle Scholar
  24. Horwitz AA et al (2015) Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas. Cell Syst 1:88–96. CrossRefPubMedGoogle Scholar
  25. Hruscha A et al (2013) Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development 140:4982–4987. CrossRefPubMedGoogle Scholar
  26. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Jinek M et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. CrossRefPubMedGoogle Scholar
  28. Johnston M, Davis RW (1984) Sequences that regulate the divergent GALJ-GALIO promoter in Saccharomyces cerevisiae. Mol Cell Biol 4:1440–1448CrossRefGoogle Scholar
  29. Joung JK, Sander JD (2013) TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14:49–55. CrossRefPubMedGoogle Scholar
  30. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Li-En Jao SRW (2013) and Wenbiao Chen efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA 110:13904–13908CrossRefGoogle Scholar
  32. Mali P et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Mans R et al (2015) CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res 15:1–15. CrossRefGoogle Scholar
  34. Miller HI (2015) Germline gene therapy: we’re ready. Science 348:1325. CrossRefPubMedGoogle Scholar
  35. Miller JC et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785. CrossRefPubMedGoogle Scholar
  36. Mukherji S, van Oudenaarden A (2009) Synthetic biology: understanding biological design from synthetic circuits. Nat Rev Genet 10:859–871. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Myers RM, Tilly K, Maniatis T (1986) Fine structure genetic analysis of a,I-G1obin promoter. Science 232:613–618CrossRefGoogle Scholar
  38. Myers RM, Lerman LS, Maniatis T (1985) A general method for saturation mutagenesis of cloned DNA fragments. Science 229:242–247CrossRefGoogle Scholar
  39. Naldini L (2015) Gene therapy returns to centre stage. Nature 526:351–360. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Packer MS, Liu DR (2015) Methods for the directed evolution of proteins. Nat Rev Genet 16:379–394. CrossRefPubMedGoogle Scholar
  41. Purnick PE, Weiss R (2009) The second wave of synthetic biology: from modules to systems. Nat Rev Mol Cell Biol 10:410–422. CrossRefPubMedGoogle Scholar
  42. Qi LS et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Renata H, Wang ZJ, Arnold FH (2015) Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution. Angew Chem 54:3351–3367. CrossRefGoogle Scholar
  44. Ritchie MD, Holzinger ER, Li R, Pendergrass SA, Kim D (2015) Methods of integrating data to uncover genotype–phenotype interactions. Nat Rev Genet 16:85–97. CrossRefPubMedGoogle Scholar
  45. Ryan OW et al (2014) Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife 3:e03703. CrossRefPubMedCentralGoogle Scholar
  46. Ryan OW, Poddar S, Cate JH (2016) CRISPR-Cas9 genome engineering in Saccharomyces cerevisiae Cells. Cold Spring Harbor protocols 2016:525–533. CrossRefGoogle Scholar
  47. SCHERER S, DAVIS RW (1979) Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci USA 76:4951–4955CrossRefGoogle Scholar
  48. Shao Z, Arnold FH (1996) Engineering new functions and altering existing functions. Curr Opin Struct Biol 6:513–518CrossRefGoogle Scholar
  49. Sharon E et al (2012) Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters. Nat Biotechnol 30:521–530. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Sharon E et al (2014) Probing the effect of promoters on noise in gene expression using thousands of designed sequences. Genome Res 24:1698–1706. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Sittig LJ et al (2016) Genetic background limits generalizability of genotype–phenotype relationships. Neuron 91:1253–1259. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Smith J et al (2000) Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 28:3361–3369CrossRefGoogle Scholar
  53. Soreanu I, Hendler A, Dahan D, Dovrat D, Aharoni A (2018) Marker-free genetic manipulations in yeast using CRISPR/CAS9 system. Curr Genet. CrossRefPubMedGoogle Scholar
  54. Stemmer M, Thumberger T, Del Sol Keyer M, Wittbrodt J, Mateo JL, CCTop (2015) An intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PloS One 10:e0124633. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Taft RJ, Pheasant M, Mattick JS (2007) The relationship between non-protein-coding DNA and eukaryotic complexity. BioEssays News Rev Mol Cell Dev Biol 29:288–299. CrossRefGoogle Scholar
  56. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636–646. CrossRefPubMedGoogle Scholar
  57. Vaschetto LM (2018) Modulating signaling networks by CRISPR/Cas9-mediated transposable element insertion. Curr Genet 64:405–412. CrossRefPubMedGoogle Scholar
  58. Wang HH et al (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460:894–898. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84. CrossRefPubMedGoogle Scholar
  60. Weinhandl K, Winkler M, Glieder A, Camattari A (2014) Carbon source dependent promoters in yeasts. Microb Cell Fact 13:1–17CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Molecular Cellular and Developmental BiologyYale UniversityNew HavenUSA
  2. 2.Systems Biology InstituteYale UniversityWest HavenUSA
  3. 3.Interdepartmental Program in Computational Biology and BioinformaticsYale UniversityNew HavenUSA
  4. 4.Department of PhysicsYale UniversityNew HavenUSA

Personalised recommendations