|Title||Incorporation of a Synthetic Amino Acid into dCas9 Improves Control of Gene Silencing|
|Author(s)||Koopal, Balwina; Kruis, Aleksander J.; Claassens, Nico J.; Nobrega, Franklin L.; Oost, John Van Der|
|Source||ACS synthetic biology 8 (2019)2. - ISSN 2161-5063 - p. 216 - 222.|
|Publication type||Refereed Article in a scientific journal|
|Keyword(s)||Cas9 - CRISPR-Cas - CRISPRi - gene silencing - synthetic amino acid|
The CRISPR-Cas9 nuclease has been repurposed as a tool for gene repression (CRISPRi). This catalytically dead Cas9 (dCas9) variant inhibits transcription by blocking either initiation or elongation by the RNA polymerase complex. Conditional control of dCas9-mediated repression has been achieved with inducible promoters that regulate the expression of the dcas9 gene. However, as dCas9-mediated gene silencing is very efficient, even slightly leaky dcas9 expression leads to significant background levels of repression of the target gene. In this study, we report on the development of optimized control of dCas9-mediated silencing through additional regulation at the translation level. We have introduced the TAG stop codon in the dcas9 gene in order to insert a synthetic amino acid, l-biphenylalanine (BipA), at a permissive site in the dCas9 protein. In the absence of BipA, a nonfunctional, truncated dCas9 is produced, but when BipA is present, the TAG codon is translated resulting in a functional, full-length dCas9 protein. This synthetic, BipA-containing dCas9 variant (dCas9-BipA) could still fully repress gene transcription. Comparison of silencing mediated by dCas9 to dCas9-BipA revealed a 14-fold reduction in background repression by the latter system. The here developed proof-of-principle system thus reduces unwanted background levels of gene silencing, allowing for tight and timed control of target gene expression.