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For double-stranded DNA (ds-DNA) repair templates, RS-1 stimulates the binding of human HDR protein RAD51 (hRAD51) to cleavage-induced single-stranded DNA (ss-DNA) over-hangs, which enhances HDR proficiency by facilitating the formation of active presynaptic filaments and stabilizing ss-DNA-RAD51 nucleoprotein filaments, without inhibiting hRAD51 ATPase activity. [ 1 ]
Note that the HDR repair of a DSB using ss-DNA as a template follows a distinct mechanism called single strand template repair (SSTR) and seems to be RAD51-indepenent. [ 2 ] Also the mechanism of RS-1 enhanced HDR via a ss-donor remains unclear. [ 3 ]
Successful Applications in CRISPR
- RS-1 improved targeted knock-in rates (from 25 to 53 %) with ss-DNA templates in (in vitro produced) bovine zygotes. [ 3 ]
- Song et al demonstrated that RS-1 enhanced Cas9- and TALEN-mediated knock-in efficiency in both in vitro and in vivo rabbit embryos via ds-DNA donors. [ 4 ]
- RS-1 treatment with ds-DNA templates was shown to enhance HDR 3- to 6-fold depending on the locus and transfection method. For instance, a 3-fold HDR enhancement was seen in human embryonic kidney (HEK293A) cells. [ 5 ]
1. Jayathilaka, K., Sheridan, S. D., Bold, T. D., Bochenska, K., Logan, H. L., Weichselbaum, R. R., Bishop, D. K., & Connell, P. P. (2008). A chemical compound that stimulates the human homologous recombination protein RAD51. Proceedings of the National Academy of Sciences of the United States of America, 105(41), 15848–15853.
2. Gallagher, D. N., Pham, N., Tsai, A. M., Janto, A. N., Choi, J., Ira, G., & Haber, J. E. (2020). A Rad51-independent pathway promotes single-strand template repair in gene editing. PLoS genetics, 16(10), e1008689.
3. Lamas-Toranzo, I., Martínez-Moro, A., O Callaghan, E., Millán-Blanca, G., Sánchez, J. M., Lonergan, P., & Bermejo-Álvarez, P. (2020). RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos. Molecular reproduction and development, 87(5), 542–549.
4. Song, J., Yang, D., Xu, J., Zhu, T., Chen, Y. E., & Zhang, J. (2016). RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nature communications, 7, 10548.
5. Pinder, J., Salsman, J., & Dellaire, G. (2015). Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Nucleic acids research, 43(19), 9379–9392.
1561729 RS-1, 98%, a RAD51 activator [CAS: 312756-74-4]