4 reasons to use TUNR instead of RNAi for your next gene knockdown experiment
TUNR is a new gene editing technology that works with CRISPR-Cas9 to allow for controlled gene knockdown. Insertion of strings of adenine nucleotides (poly A tracks) into the coding region of a gene results in a proportional level of knockdown—long polyA tracks produce a strong knockdown of gene expression, whereas shorter polyA tracks generate a more modest effect.
RNA interference (RNAi) is a technology similar to TUNR in that it causes gene knockdown. RNAi has limitations, however, and here are the top 4 reasons to use TUNR instead of RNAi:
TUNR generates multiple predictable levels of knockdown
Small interfering- and short hairpin RNAs (si- and shRNA, respectively) generate unpredictable levels of knockdown. To achieve a specific level of knockdown with si- and shRNA, multiple constructs (sometimes dozens!) must be tested in a trial-and-error methodology to find useable reagents. TUNR is much more predictable- the longer the poly-Lysine track, the greater the knockdown.
Knockdown by TUNR is permanent and heritable
The effects of siRNA are transient—the siRNA is active, and then degraded and removed from the cell, allowing expression levels of the target gene to return to normal. Stable cell lines can be generated with shRNA, however this is the result of random integration of the shRNA into the genome with the potential to disrupt another gene. Additionally, expression levels of the shRNA may change over time, and must be monitored.
TUNR, however, results in knockdown that is permanent, stable, and heritable. The TUNR sequence is integrated in a targeted fashion, directly into the endogenous gene. As such, fluctuations in copy number are rare, and expression is stable and heritable.
TUNR has few off target effects
Off target effects of sh- and siRNA are well documented—mismatches are tolerated and can lead to knockdown of genes other than the target gene that have some degree of complement with the siRNA. In order to be certain the observed phenotype is not due to off target activity, researchers will commonly utilize two or three siRNAs to the same target. Additionally, transfection alone can cause the induction or repression of other genes, and may also generate an immune response.
Lastly, random integration of shRNA may result in disruption of off target genes. These problems are avoided with TUNR. TUNR relies on disruption of translation induced by the inserted polyA tracks, and therefore hybridization with incorrect targets is not of issue.
Additionally, insertion of TUNR sequences is done in a controlled and targeted manner, and sequencing is performed to demonstrate proper insertion—disruption of off target genes via random insertion is again not of issue. Lastly, transfection effects and immunogenicity are not of concern as the TUNR effect is not transient and the cells can be allowed to normalize following TUNR insertion.
TUNR expression is intrinsically linked with gene target expression
Expression of shRNA constructs is dependent on the promoter driving the shRNA. Some commonly used promoters suffer from weak expression in certain cell types—e.g. CMV expresses poorly in neuronal cells.
Conversely, TUNR is driven off the same promoter driving the endogenous target gene. This ensures TUNR activity in relevant cell types.