Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

Abstract

A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks which are then repaired by homology-directed repair using DNA donor molecules carrying desired mutations. Using a mixture of small molecules, the “CRISPY” mix, we achieve a 2.8- to 7.2-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes or of gene encoding a blue fluorescent protein in 27% of cells, to our knowledge the highest editing efficiency in human induced pluripotent stem cells described to date. Furthermore, the CRISPY mix improves precise genome editing with Cpf1 2.3- to 4.0-fold, allowing almost 20% of chromosomes to be edited. The components of the CRISPY mix do not always increase the editing efficiency in the immortalized or primary cell lines tested, suggesting that employed repair pathways are cell-type specific.

References 49

1. 1.

   Robinton DA, Daley GQ. The promise of induced pluripotent stem cells in research and therapy. Nature. 2012;481:295-305.

   Not in our library10.1038/nature10761

2. 2.

   Mandai M, Kurimoto Y, Takahashi M. Autologous induced stem-cell-derived retinal cells for macular degeneration. N. Engl. J. Med. 2017;376:1038-1046.

   Not in our library10.1056/nejmoa1608368

3. 3.

   Shrivastav M, De Haro LP, Nickoloff JA. Regulation of DNA double-strand break repair pathway choice. Cell Res. 2008;18:134-147.

   Not in our library10.1038/cr.2007.111

4. 4.

   Dueva R, Iliakis G. Alternative pathways of non-homologous end joining (NHEJ) in genomic instability and cancer. Transl. Cancer Res. 2013;2:163-177.

   Not in our library

5. 5.

   Nussenzweig A, Nussenzweig MC. A backup DNA repair pathway moves to the forefront. Cell. 2007;131:223-225.

   Not in our library10.1016/j.cell.2007.10.005

6. 6.

   Grimme JM, et al. Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52-ssDNA complexes. Nucleic Acids Res. 2010;38:2917-2930.

   Not in our library10.1093/nar/gkp1249

7. 7.
   Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus · 2017

   No notice
8. 8.

   Fu Y, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 2013;31:822-826.

   Not in our library10.1038/nbt.2623

9. 9.

   Shen B, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods. 2014;11:399-402.

   Not in our library10.1038/nmeth.2857

10. 10.

    Zetsche B, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163:759-771.

    Not in our library10.1016/j.cell.2015.09.038

11. 11.

    Kim D, et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 2016;34:863-868.

    Not in our library10.1038/nbt.3609

12. 12.

    Kleinstiver BP, et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 2016;34:869-874.

    Not in our library10.1038/nbt.3620

13. 13.

    Gonzalez F, et al. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell. 2014;15:215-226.

    Not in our library10.1016/j.stem.2014.05.018

14. 14.

    Yu C, et al. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell. 2015;16:142-147.

    Not in our library10.1016/j.stem.2015.01.003

15. 15.
    Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery · 2014

    No notice
16. 16.
    Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases · 2016

    No notice
17. 17.

    Chu VT, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 2015;33:543-548.

    Not in our library10.1038/nbt.3198

18. 18.

    Maruyama T, et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 2015;33:538-542.

    Not in our library10.1038/nbt.3190

19. 19.

    Singh P, Schimenti JC, Bolcun-Filas E. A mouse geneticist’s practical guide to CRISPR applications. Genetics. 2015;199:1-15.

    Not in our library10.1534/genetics.114.169771

20. 20.
    Nuclear domain ‘knock-in’ screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing · 2015

    No notice
21. 21.
    RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency · 2016

    No notice
22. 22.
    Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage · 2017

    Corrected
23. 23.

    Greco GE, et al. SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV. DNA Repair. 2016;43:18-23.

    Not in our library10.1016/j.dnarep.2016.04.004

24. 24.
    Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing · 2015

    No notice
25. 25.

    Suzuki K, et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature. 2016;540:144-149.

    Not in our library10.1038/nature20565

26. 26.

    Wang K, et al. Efficient generation of orthologous point mutations in pigs via CRISPR-assisted ssODN-mediated homology-directed repair. Mol. Ther. Nucleic Acids. 2016;5:e396.

    Not in our library10.1038/mtna.2016.101

27. 27.

    Milanowska K, et al. REPAIRtoire–a database of DNA repair pathways. Nucleic Acids Res. 2011;39:D788-D792.

    Not in our library10.1093/nar/gkq1087

28. 28.
    The ChEMBL bioactivity database: an update · 2013

    No notice
29. 29.

    O’Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 2000;267:5421-5426.

    Not in our library10.1046/j.1432-1327.2000.01606.x

30. 30.
    An Enhanced Monomeric Blue Fluorescent Protein with the High Chemical Stability of the Chromophore · 2011

    No notice
31. 31.

    Lee JS. Activation of ATM-dependent DNA damage signal pathway by a histone deacetylase inhibitor, trichostatin A. Cancer Res. Treat. 2007;39:125-130.

    Not in our library10.4143/crt.2007.39.3.125

32. 32.

    Jimeno S, et al. Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice. Nucleic Acids Res. 2015;43:987-999.

    Not in our library10.1093/nar/gku1384

33. 33.

    Glanzer JG, Liu S, Oakley GG. Small molecule inhibitor of the RPA70 N-terminal protein interaction domain discovered using in silico and in vitro methods. Bioorg. Med. Chem. 2011;19:2589-2595.

    Not in our library10.1016/j.bmc.2011.03.012

34. 34.

    Glanzer JG, et al. A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A. Nucleic Acids Res. 2013;41:2047-2059.

    Not in our library10.1093/nar/gks1291

35. 35.

    Blanpain C, Mohrin M, Sotiropoulou PA, Passegue E. DNA-damage response in tissue-specific and cancer stem cells. Cell Stem Cell. 2011;8:16-29.

    Not in our library10.1016/j.stem.2010.12.012

36. 36.

    Rocha CR, Lerner LK, Okamoto OK, Marchetto MC, Menck CF. The role of DNA repair in the pluripotency and differentiation of human stem cells. Mutat. Res. 2013;752:25-35.

    Not in our library10.1016/j.mrrev.2012.09.001

37. 37.

    Gomez-Cabello D, Checa-Rodriguez C, Abad M, Serrano M, Huertas P. CtIP-specific roles during cell reprogramming have long-term consequences in the survival and fitness of induced pluripotent stem cells. Stem Cell Rep. 2017;8:432-445.

    Not in our library10.1016/j.stemcr.2016.12.009

38. 38.
    Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development · 2016

    No notice
39. 39.

    Prüfer K, et al. The complete genome sequence of a Neanderthal from the Altai Mountains. Nature. 2014;505:43-49.

    Not in our library10.1038/nature12886

40. 40.

    Chari R, Mali P, Moosburner M, Church GM. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat. Methods. 2015;12:823-826.

    Not in our library10.1038/nmeth.3473

Cited by papers we hold 9

• Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing · 2023
  No notice
• Inferring and perturbing cell fate regulomes in human brain organoids · 2022
  No notice
• Lineage recording in human cerebral organoids · 2021
  No notice
• Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing · 2021
  No notice
• Synergistic gene editing in human iPS cells via cell cycle and DNA repair modulation · 2020
  Not checked
• Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook · 2020
  No notice
• Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks · 2020
  No notice
• CRISPR Cpf1 proteins: structure, function and implications for genome editing · 2019
  No notice
• Simultaneous precise editing of multiple genes in human cells · 2019
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.