Advanced search
1 file | 3.83 MB

CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments

Annekatrien Boel (UGent) , Hanna De Saffel (UGent) , Wouter Steyaert (UGent) , Bert Callewaert (UGent) , Anne De Paepe (UGent) , Paul Coucke (UGent) and Andy Willaert (UGent)
Author
Organization
Abstract
Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.
Keywords
CRISPR/Cas9, HDR, Homology-directed repair, Zebrafish, Next-generation sequencing, STRAND-BREAK REPAIR, ILLEGITIMATE DNA INTEGRATION, KNOCK-IN, HIGH-THROUGHPUT, CRISPR, RNA, EFFICIENCY, RECOMBINATION, CAS9, GENERATION

Downloads

  • dmm035352.full.pdf
    • full text
    • |
    • open access
    • |
    • PDF
    • |
    • 3.83 MB

Citation

Please use this url to cite or link to this publication:

Chicago
Boel, Annekatrien, Hanna De Saffel, Wouter Steyaert, Bert Callewaert, Anne De Paepe, Paul Coucke, and Andy Willaert. 2018. “CRISPR/Cas9-mediated Homology-directed Repair by ssODNs in Zebrafish Induces Complex Mutational Patterns Resulting from Genomic Integration of Repair-template Fragments.” Disease Models & Mechanisms 11 (10).
APA
Boel, A., De Saffel, H., Steyaert, W., Callewaert, B., De Paepe, A., Coucke, P., & Willaert, A. (2018). CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments. DISEASE MODELS & MECHANISMS, 11(10).
Vancouver
1.
Boel A, De Saffel H, Steyaert W, Callewaert B, De Paepe A, Coucke P, et al. CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments. DISEASE MODELS & MECHANISMS. 2018;11(10).
MLA
Boel, Annekatrien et al. “CRISPR/Cas9-mediated Homology-directed Repair by ssODNs in Zebrafish Induces Complex Mutational Patterns Resulting from Genomic Integration of Repair-template Fragments.” DISEASE MODELS & MECHANISMS 11.10 (2018): n. pag. Print.
@article{8602691,
  abstract     = {Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4\% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.},
  articleno    = {dmm035352},
  author       = {Boel, Annekatrien and De Saffel, Hanna and Steyaert, Wouter and Callewaert, Bert and De Paepe, Anne and Coucke, Paul and Willaert, Andy},
  issn         = {1754-8403},
  journal      = {DISEASE MODELS \& MECHANISMS},
  language     = {eng},
  number       = {10},
  pages        = {11},
  title        = {CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments},
  url          = {http://dx.doi.org/10.1242/dmm.035352},
  volume       = {11},
  year         = {2018},
}

Altmetric
View in Altmetric
Web of Science
Times cited: