What are additional methods for improving HDR efficiency?
What are additional methods for improving HDR efficiency?
The development of novel methods for improving HDR efficiency is currently a major focus area in the genome editing field. Toward this end, the following approaches have shown promise:
-Fusion or attachment of Cas9-sgRNA RNP and template (Aird et al. 2018; Savic et al. 2018; Carlson-Stevermer et al. 2017)
-Restriction of Cas9 expression to S and G2 phases of the cell cycle, when the propensity for HDR is highest (Gutschner et al. 2016)
-Development of base editing for engineering single nucleotide substitutions (Rees and Liu 2018)
-Application of various chemical compounds that increase the percentage of HDR (Wienert et al. 2018; Riesenberg and Maricic 2018)
References
Aird, E. J., Lovendahl, K. N., St. Martin, A., Harris, R. S. & Gordon, W. R. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun. Biol. 1, 54 (2018).
Carlson-Stevermer, J. et al. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat. Commun. 8, 1711 (2017).
Gutschner, T., Haemmerle, M., Genovese, G., Draetta, G. F. & Chin, L. Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair. Cell Rep. 14, 1555–1566 (2016).
Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).
Riesenberg, S. & Maricic, T. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. Nat. Commun. 9, 2164 (2018).
Savic, N. et al. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. Elife 7, (2018).
Wienert, B. et al. Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair. bioRxiv 500462 (2018). doi:10.1101/500462