The ability of a single Cas9 protein to regulate RNA production while also maintaining the capacity to cleave DNA will be of great use in deciphering complex biological interactions and developing artificial genetic circuits. A promising use of our gRNA design principles will be in easily extending existing Cas9-based genome editing systems to concurrently modulate gene expression. This is particularly appealing in cases where considerable effort has been expended toward the generation of Cas9-expressing strains of mice or other labor-intensive and costly model systems13,14. Further, our data suggest that nuclease-positive Cas9 can be easily endowed with other previously described dCas9 activities15,16 such as in vivo chromosomal tracking17, and they could facilitate the development of multifunctional synthetic genetic safety circuits with potential
biomedical applications.