The development of a controllable Cas9 system that is chemically inducible would greatly enhance the experimental utility of the CRISPR Cas9 system.
In the CRISPR Cas9 system, a guide RNA directs the Cas9 endonuclease to a specific region within the genome where it introduces a double stranded break in the DNA. Various modifications of the Cas9 protein have been made, including changing the Cas9 protein so that it introduces single stranded nicks, as well as completely inactivating Cas9 and fusing with other proteins to modulate gene transcription.
In an advance online publication in Nature Biotechnology, Zetsche et al (2015) describe an innovative use of the RNA-guided CRISPR Cas9 system, where they split the Cas9 protein into N- and C-termini and fused them to the FK506 binding protein 12 (FKBP) and the FKBP rapamycin binding (FRB) domains of mTOR, respectively. This system, when co-expressed in the presence of rapamycin, restores the activity of the Cas9 protein.
To identify how to “split” the protein, Zetsche et al used the crystal structure of Cas9 and identified two positions where the Cas9 could be divided. Based on this, they developed a single cassette, with which they transfected HEK293FT cells, and demonstrated that Cas9-mediated cleavage occurred only in the presence of rapamycin.
In addition, the split Cas9 system was found to be more precise than wild type Cas9, which the authors demonstrated by transducing HEK293FT cells with wild type Cas9 or with the split Cas9 system, which showed that the split system generated fewer off-target indels than wild type Cas9, likely due to the transcient activity of the split system, whereas the prolonged exposure to wild type Cas9 led to off target indels .
Finally, Zetsche et al tested the utility of the split Cas9 system with an inactivated dCas9 with the C-terminus fused to the VP64 activation domain, and demonstrated that they could, in fact, induce gene expression, which resulted in persistent activation following rapamycin withdrawl (Figure 4).
For future uses and directions, the authors mention that the system could be useful for restricting Cas9 activity to specific cells through the use of tissue-specific promoters, and also mention that there is the potential for the system to be used with other chemically inducible dimerization domains.