The DOI matches its Crossref record; 22 of the 22 references it cites were found in a registry or an index.
A Survey of Validation Strategies for CRISPR-Cas9 Editing
Open accessAbstract
The T7 endonuclease 1 (T7E1) mismatch detection assay is a widely used method for evaluating the activity of site-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. To determine the accuracy and sensitivity of this assay, we compared the editing estimates derived by the T7E1 assay with that of targeted next-generation sequencing (NGS) in pools of edited mammalian cells. Here, we report that estimates of nuclease activity determined by T7E1 most often do not accurately reflect the activity observed in edited cells. Editing efficiencies of CRISPR-Cas9 complexes with similar activity by T7E1 can prove dramatically different by NGS. Additionally, we compared editing efficiencies predicted by the Tracking of Indels by Decomposition (TIDE) assay and the Indel Detection by Amplicon Analysis (IDAA) assay to that observed by targeted NGS for both cellular pools and single-cell derived clones. We show that targeted NGS, TIDE, and IDAA assays predict similar editing efficiencies for pools of cells but that TIDE and IDAA can miscall alleles in edited clones.
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Cited by
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Conant, D., Hsiau, T., Rossi, N., Oki, J., Maures, T., Waite, K., Yang, J., Joshi, S., Kelso, R., Holden, K., et al. (2022) · The CRISPR Journal · doi:10.1089/crispr.2021.0113 · Cited by 804
Evaluating and enhancing target specificity of gene-editing nucleases and deaminases
Kim, D., Luk, K., Wolfe, S. A., & Kim, J.-S. (2019) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-013118-111730 · Cited by 171
Gene correction for SCID-X1 in long-term hematopoietic stem cells
Pavel-Dinu, M., Wiebking, V., Dejene, B. T., Srifa, W., Mantri, S., Nicolas, C. E., Lee, C., Bao, G., Kildebeck, E. J., Punjya, N., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-09614-y · Cited by 167
Bloh, K., Kanchana, R., Bialk, P., Banas, K., Zhang, Z., Yoo, B.-C., & Kmiec, E. B. (2021) · The CRISPR Journal · doi:10.1089/crispr.2020.0022 · Cited by 165
Izmiryan, A., Ganier, C., Bovolenta, M., Schmitt, A., Mavilio, F., & Hovnanian, A. (2018) · Molecular Therapy Nucleic Acids · doi:10.1016/j.omtn.2018.06.008 · Cited by 68
Metadata
- Authors
- Sentmanat, M. F., Peters, S. T., Florian, C. P., Connelly, J. P., & Pruett-Miller, S. M.
- Venue
- Scientific Reports
- Published
- 2018-01-17
- Volume
- 8
- Issue
- 1
- Pages
- 888-888
- ISSN
- 2045-2322 (ISSN-L)
- OpenAlex
- W2783501849
- Cited by
- 341
- References
- 22
- FWCI
- 10.91
- Type
- journal article
- Language
- en
- Open access
- gold
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