A recent manuscript by Suzuki et al (2018) demonstrates an effective method to control the expression of CRISPR Cas9 based molecular components with a small molecule.
Existing methods for controlling Cas9 suffer from high background activity and/or use compounds such as antibiotics or pharmaceuticals, which may not be acceptable for in situ control outside of the laboratory. Suzuki et al sought to address these limitations by using H-Lys(Boc)-OH (BOC); a lysine derivative which is incorporated at UAGs (amber stop codon) by pyrrolysyl-tRNA.
By replacing lysine codons with UAGs, the translation of full length proteins becomes dependent on the availability of BOC, pyrrolysyl-tRNA synthetase, and pyrrolysyl-tRNA.
This system was first tested in mouse embryos by co-injecting oocytes with sperm and plasmids encoding the pyrrolysyl-tRNA synthetase/tRNA along with eGFP containing a premature amber stop codon at position 150. Robust eGFP expression was only detected when the resulting embryos were incubated with BOC.
Next, the authors tested how well this approach can be used to regulate Cas9 activity in HEK293 cells and mouse embryos. Plasmids encoding pyrrolysyl-tRNA synthetase/tRNA, eGFP targeting sgRNA, along with Cas9 variants where lysine codons 510, 742, or both combined are replaced with UAGs where tranfected into HEK293 cells along with an eGFP plasmid or co-injected into mouse oocytes with sperm containing an eGFP transgene. In the absence of BOC, eGFP was detected while full length Cas9 was not. In the presence of BOC, full length Cas9 was present and effectively targeted eGFP coding DNA and suppressed eGFP expression.
The authors also targeted native genes in mouse embryos whose mutation would lead to an observable phenotype followed by implanting them into surrogate mothers. The absence of BOC resulted in no pups with the mutant allele phenotype; incubating the embryos with BOC prior to implantation produced mutant phenotype pups at a rate comparable to wild-type Cas9 controls.
This paper demonstrates the possibility of using translational control with an orthogonal tRNA synthetase/tRNA pair and an exogenously supplied non-standard amino acid. Importantly, the authors demonstrated that this approach results in little to no background activity.
If BOC can effectively regulate systemic Cas9 activity when supplied through the diet, this could be applied to insect systems to control life stage specific genome editing or transcriptional control. Limiting Cas9 activity via access to BOC may also hold promise as a biocontainment system for gene-drive research and potentially regulate the activity of Cas9 based biocontrol systems in the wild.
Toru Suzuki, Maki Asami, Sanjay G. Patel, Louis Y. P. Luk, Yu-Hsuan Tsai & Anthony C. F. Perry (2018). Switchable genome editing via genetic code expansion. Scientific Reports 8, Article number: 10051