Cas9 excision of the DNA sequence of interest depends on guide RNA (gRNA) and protospacer adjacent motif (PAM). Recall that gRNA directs Cas9 to the site and PAM pinpoints precisely where DNA excision should take place.
One way of minimizing the CRISPR/Cas9 off-target effect and maximizing on-target DNA cleavage is to optimize the rational design of gRNA, that is, RNA sequence that is complementary to the targeted DNA sequence.36 The aim is to predict optimal gRNA-guided Cas9 DNA excision of intended sequences; gRNA design is a developing area of research in bioinformatics.
36. Perez, A. R., Pritykin, Y., Vidigal, J. A., Chhangawala, S., Zamparo, L., Leslie, C. S., & Ventura, A. (2017). GuideScan software for improved single and paired CRISPR guide RNA design. Nature Biotechnology, 35(4), 347–349. https://doi.org/10.1038/nbt.3804
