Letter | Published:

Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9

Nature volume 533, pages 125129 (05 May 2016) | Download Citation

Abstract

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells1,2. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels)2. DSBs may also be repaired by homology-directed repair (HDR)1,2 using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations3. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient3,4,5,6,7,8 and can be corrupted by additional indels9, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed ‘CORRECT’ for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation’s incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer’s disease-causing mutations in amyloid precursor protein (APPSwe)10 and presenilin 1 (PSEN1M146V)11 and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–1278 (2013)

  2. 2.

    , & Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014)

  3. 3.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

  4. 4.

    et al. Inducible in vivo genome editing with CRISPR-Cas9. Nature Biotechnol. 33, 390–394 (2015)

  5. 5.

    et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014)

  6. 6.

    et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013)

  7. 7.

    et al. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J. Biol. Chem. 289, 21312–21324 (2014)

  8. 8.

    et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013)

  9. 9.

    et al. Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system. Sci. Rep. 4, 5396 (2014)

  10. 10.

    et al. The Swedish mutation causes early-onset Alzheimer’s disease by beta-secretase cleavage within the secretory pathway. Nature Med. 1, 1291–1296 (1995)

  11. 11.

    Alzheimer’s Disease Collaborative Group. The structure of the presenilin 1 (S182) gene and identification of six novel mutations in early onset AD families. Nature Genet. 11, 219–222 (1995)

  12. 12.

    , , , & RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnol. 31, 233–239 (2013)

  13. 13.

    et al. Optimization of scarless human stem cell genome editing. Nucleic Acids Res. 41, 9049–9061 (2013)

  14. 14.

    et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015)

  15. 15.

    , , & Regulation of gene editing activity directed by single-stranded oligonucleotides and CRISPR/Cas9 systems. PLoS ONE 10, e0129308 (2015)

  16. 16.

    , , , & Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cell. Biol. 18, 93–101 (1998)

  17. 17.

    , , & Donor DNA utilization during gene targeting with zinc-finger nucleases. Genes Genomes Genetics (G3) 3, 657–664 (2013)

  18. 18.

    , , , & The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides. PLoS ONE 9, e96483 (2014)

  19. 19.

    & Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells. Mol. Cell. Biol. 17, 6386–6393 (1997)

  20. 20.

    & Animal models of Alzheimer’s disease and frontotemporal dementia. Nature Rev. Neurosci. 9, 532–544 (2008)

  21. 21.

    et al. Modeling familial Alzheimer’s disease with induced pluripotent stem cells. Hum. Mol. Genet. 20, 4530–4539 (2011)

  22. 22.

    et al. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells. Nature 482, 216–220 (2012)

  23. 23.

    et al. Modeling Alzheimer’s disease with iPSCs reveals stress phenotypes associated with intracellular Aβ and differential drug responsiveness. Cell Stem Cell 12, 487–496 (2013)

  24. 24.

    et al. The familial Alzheimer’s disease APPV717I mutation alters APP processing and Tau expression in iPSC-derived neurons. Hum. Mol. Genet. 23, 3523–3536 (2014)

  25. 25.

    et al. Characterization and molecular profiling of PSEN1 familial Alzheimer’s disease iPSC-derived neural progenitors. PLoS ONE 9, e84547 (2014)

  26. 26.

    et al. The presenilin-1 ΔE9 mutation results in reduced γ-secretase activity, but not total loss of PS1 function, in isogenic human stem cells. Cell Rep. 5, 974–985 (2013)

  27. 27.

    , , & Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2015)

  28. 28.

    et al. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16, 142–147 (2015)

  29. 29.

    et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nature Biotechnol. 33, 538–542 (2015)

  30. 30.

    et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nature Biotechnol. 33, 543–548 (2015)

  31. 31.

    et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnol. 31, 822–826 (2013)

  32. 32.

    et al. Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting. PLoS ONE 8, e59867 (2013)

  33. 33.

    et al. Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350, 1353–1356 (2004)

  34. 34.

    , & & The Galaxy Team. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 11, R86 (2010)

  35. 35.

    et al. Galaxy: a web-based genome analysis tool for experimentalists. Curr. Protoc. Mol. Biol. Chapter 19, Unit 19.10.1–21 (2010)

  36. 36.

    FastQC: a quality control tool for high throughput sequence data. ()

  37. 37.

    , , & PEAR: a fast and accurate Illumina paired-end read merger. Bioinformatics 30, 614–620 (2014)

  38. 38.

    , , & Comparison of DNA sequences with protein sequences. Genomics 46, 24–36 (1997)

  39. 39.

    et al. Manipulation of FASTQ data with Galaxy. Bioinformatics 26, 1783–1785 (2010)

  40. 40.

    & Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010)

  41. 41.

    et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nature Biotechnol. 32, 569–576 (2014)

  42. 42.

    , , , & Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nature Biotechnol. 32, 267–273 (2014)

  43. 43.

    R Core Team. R: A language and environment for statistical computing. (R Foundation for Statistical Computing, 2015)

  44. 44.

    , & The iCRISPR platform for rapid genome editing in human pluripotent stem cells. Methods Enzymol. 546, 215–250 (2014)

  45. 45.

    et al. Poly peak parser: method and software for identification of unknown indels using Sanger sequencing of polymerase chain reaction products. Dev. Dyn. 243, 1632–1636 (2014)

  46. 46.

    , , , & COSMID: a web-based tool for identifying and validating CRISPR/Cas off-target sites. Mol. Ther. Nucleic Acids 3, e214 (2014)

  47. 47.

    , & Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nature Protocols 7, 1836–1846 (2012)

  48. 48.

    et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013)

  49. 49.

    et al. A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline. Nature 488, 96–99 (2012)

Download references

Acknowledgements

This research was supported by The Rockefeller University, The New York Stem Cell Foundation, The Ellison Foundation, Cure Alzheimer’s Fund, the Empire State Stem Cell fund through new York State Department of Health contract number C023046, and CTSA, RUCCTS grant number 8 UL1 TR000043 from the National Center for Advancing Translational Sciences (NCATS, NIH). D.P. is a New York Stem Cell Foundation Druckenmiller Fellow and received a fellowship from the German Academy of Sciences Leopoldina. D.K. is a Howard Hughes Medical Institute International Student Research Fellow and received a fellowship from the National Sciences and Engineering Research Council of Canada. S.T. is supported by the Agency for Science, Technology and Research of Singapore. A.G. is supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-institutional MD-PhD program. We thank members of the Tessier-Lavigne laboratory and L. Marraffini for discussions. Our thanks also go to S. Mazel and the team at the Rockefeller Univeristy Flow Cytometry Resource Center, J. Gonzalez and the team at the Rockefeller University Translational Technology Core Laboratory, C. Zhao and the team at the Rockefeller University Genomics Resource Center, and D. Paull and M. Duffield for technical help. Opinions expressed here are solely those of the authors and do not necessarily reflect those of the Empire State Stem Cell Fund, the New York State Department of Health, or the State of New York. The content of this study is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Author notes

    • Dominik Paquet
    •  & Dylan Kwart

    These authors contributed equally to this work.

    • Andrew Sproul

    Present address: Department of Pathology and Cell Biology and the Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University Medical Center, 630 West 168th Street, New York, New York 10032, USA.

Affiliations

  1. Laboratory of Brain Development and Repair, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA

    • Dominik Paquet
    • , Dylan Kwart
    • , Antonia Chen
    • , Shaun Teo
    • , Kimberly Moore Olsen
    • , Andrew Gregg
    •  & Marc Tessier-Lavigne
  2. The New York Stem Cell Foundation Research Institute, New York, New York 10032, USA

    • Andrew Sproul
    • , Samson Jacob
    •  & Scott Noggle
  3. Weill Cornell Graduate School of Medical Sciences, The Rockefeller University and Sloan-Kettering Institute Tri-institutional MD-PhD Program, 1300 York Avenue, New York, New York 10065, USA

    • Andrew Gregg

Authors

  1. Search for Dominik Paquet in:

  2. Search for Dylan Kwart in:

  3. Search for Antonia Chen in:

  4. Search for Andrew Sproul in:

  5. Search for Samson Jacob in:

  6. Search for Shaun Teo in:

  7. Search for Kimberly Moore Olsen in:

  8. Search for Andrew Gregg in:

  9. Search for Scott Noggle in:

  10. Search for Marc Tessier-Lavigne in:

Contributions

D.P., D.K. and M.T.-L. conceived and designed the study. D.P. and D.K. performed and analysed the experiments. A.C. and A.G. helped perform the experiments. S.T. helped analyse next-generation sequencing data. A.S., S.J. and S.N. generated and characterized the iPS cells. K.M.O. performed and analysed the electrophysiology assays. D.P., D.K., and M.T.-L. wrote the manuscript with input from all authors.

Competing interests

A patent application relating to this work has been filed by D.P., D.K. and M.T.-L.

Corresponding author

Correspondence to Marc Tessier-Lavigne.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Data

    This file contains Supplementary Table 1, a list of APPSwe / PSEN1M146V sgRNAs and primers used for amplification of targeted loci.

  2. 2.

    Supplementary Data

    This file contains Supplementary Table 2, a list of ssODNs used as repair templates and enzymes used for RFLP analysis.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature17664

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.