Two additional and highly recommended approaches include:37
- Minimizing the CRISPR/Cas9 off-target effect by relying on highly purified Cas9 protein, which expedites the time taken for CRISPR/Cas9 to take effect, hence narrowing the temporal window for off-targeting phenomenon to occur.
- Maximizing on-target delivery by selecting an alternative DNA excision molecule (also called DNA nuclease). Cas9 is the first-studied CRISPR-associated DNA nuclease of relatively large size. Size could serve as an obstacle to efficient CRISPR delivery to cells and can contribute to off-target effects. Pioneered by Dr. Deng Zhang’s laboratory at the Broad Institute, co-run by Harvard University and Massachusetts Institute of Technology (MIT), a more optimal DNA nuclease has been born, termed Cpf1, also called Cas12a, which bears several advantages compared to Cas9. Cpf1 is smaller in size which renders it easier to be delivered to mammalian cells for therapeutic studies.
37. Morgens, D. W., Deans, R. M., Li, A., & Bassik, M. C. (2016). Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nature biotechnology, 34(6), 634–636. https://doi.org/10.1038/nbt.3567
