The DOI matches its Crossref record; 66 of the 66 references it cites were found in a registry or an index.
Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage
Open accessAbstract
Background: Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs). Results: Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97–100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs. Conclusions: Taken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.
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Xue, C., & Greene, E. C. (2021) · Trends in Genetics · doi:10.1016/j.tig.2021.02.008 · Cited by 431
The emerging and uncultivated potential of CRISPR technology in plant science
Zhang, Y., Malzahn, A. A., Sretenovic, S., & Qi, Y. (2019) · Nature Plants · doi:10.1038/s41477-019-0461-5 · Cited by 394
RNA–protein interaction detection in living cells
Ramanathan, M., Majzoub, K., Rao, D. S., Neela, P. H., Zarnegar, B. J., Mondal, S., Roth, J. G., Gai, H., Kovalski, J. R., Siprashvili, Z., et al. (2018) · Nature Methods · doi:10.1038/nmeth.4601 · Cited by 308
Yang, H., Ren, S., Yu, S., Pan, H., Li, T., Ge, S., Zhang, J., & Xia, N. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21186461 · Cited by 237
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Metadata
- Authors
- Zhang, J.-P., Li, X.-L., Li, G.-H., Chen, W., Arakaki, C., Botimer, G. D., Baylink, D., Zhang, L., Wen, W., Fu, Y.-W., Xu, J., Chun, N., Yuan, W., Cheng, T., & Zhang, X.-B.
- Venue
- Genome Biology
- Published
- 2017-02-20
- Volume
- 18
- Issue
- 1
- Pages
- 35-35
- ISSN
- 1465-6906 (ISSN-L)
- 1474-760X
- OpenAlex
- W2590493137
- Cited by
- 478
- References
- 66
- FWCI
- 16.99
- Type
- journal article
- Language
- en
- Open access
- gold
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- Zhang, J.-P., Li, X.-L., Li, G.-H., Chen, W., Arakaki, C., Botimer, G. D., Baylink, D., Zhang, L., Wen, W., Fu, Y.-W., Xu, J., Chun, N., Yuan, W., Cheng, T., & Zhang, X.-B. (2017). Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biology. https://doi.org/10.1186/s13059-017-1164-8
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