RNAi offer >70% knock-down of the gene expression, and thanks to CRISPR-CAS9 gene editing, it’s possible make a complete knock-out. The TUNR system offers the possibility to obtain intermediates with high, medium and low reduction of the gene expression and with the same quality of targeting as for knock-out. Let’s take a closer look at the TUNR technique.
Gene Editing: Wild-type, knockout, and everywhere in between
The TUNR technique is based on the insertion of a polyA sequence into the target gene. Depending on the length of that TUNR sequence , the level of the protein expression will be more or less reduced. Thus, the gene expression level can be precisely controlled. It offers intermediates between wild type and knock-out to decipher a function or for a component screening such as for synthetic lethality.
- Precisely tune gene expression from 100% all the way down to complete knockout
- Model ranges of gene expression reflecting diversity in patient populations
- Control endogenous gene expression–eliminate artifacts from ectopic expression
- Knockdown essential genes where knockout would result in lethality
A CRISPR-CAS9 based targeting
The TUNR gene editing system leverages the CRISPR-Cas9 system for targeted insertion of polyA tracks into a precise location within the target gene.
Control the expression level
Insertion of tracks of repeated adenine bases results in decreased mRNA and protein expression. PolyA track length is inversely proportional to protein expression.
The picture is adapted from a recent Nature communication: Arthur, L. L. et al. Rapid generation of hypomorphic mutations. Nat.Commun. 8, 14112 doi:10.1038/ncom-ms14112 (2017).
Case study, PolyA tracks reduce protein expression of mCherry in HeLa cells
Insertion of polyA tracks in an mCherry construct reduces protein expression in a dose-dependent manner. Insertion of 12 AAA codons (36 adenosines, “TUNR-H”) results in the highest level of reduction, followed by (AAA)9 (TUNR-M) and then (AAA)6 (TUNR-L). Insertion of the alternative lysine codon AAG did not affect protein expression. Top panel: quantification of western blot. Lower panel: western blot.
Take it easy – one single box per project
There are 3 kinds of projects:
- Tune the endogenous expression of a gene, TUNR0008 kit
- Insert in the AAVS1 Safe-harbor kit a transgene with the TUNR system, TUNR0005 kit– it is for human cell line establishment
- Add a function at different level with TUNR expression plasmid, TUNR0001 kit– it could be transient expression or with random insertions.
For each project just order the preferred kit mentioning the gene ID.
And for complete peace of mind for your projects requiring gene editing, why not ask a quote for our services mentioning your target gene ID and cells.
