WEBINAR - TUNR Flexible Gene Editing System

WEBINAR - TUNR Flexible Gene Editing System

"Targeted Insertion of PolyA Tracks with CRISPR-Cas9 Allows Titratable Control of Gene Expression" Speaker: Rachel Delston, PhD, Canopy Biosciences (May 3rd 2018).

Watch the Webinar here.

CRISPR Gene Expression Control System

CRISPR Gene Expression Control System
TUNR Flexible Gene Editing System

TUNR Flexible Gene Editing System

Control your Gene Expression by insertion of small polyA tracks in your ORF... 

  • 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.

Validated in:

  • Human cells
  • Plants
  • Bacteria
  • Yeast
  • Protozoans
  • Insects
  • ...

 

3 Custom Kits Available:

 Endogenous Genes

  • The TUNR Flexible Gene Editing System for Endogenous Genes allows you to generate four cell lines or animal models with four different levels of knockdown of your endogenous gene of interest (more Information about the kit components).

Targeted Transgenes

  • The TUNR Targeted Transgenic kit enables you to insert your gene of interest under TUNR control into the AAVS1 locus. AAVS1 is a “safe harbor”—a safe place to integrate transgenes, allowing predictable expression from a locus known not to alter the expression of other genes or have otherwise deleterious effects on the cell (more information about the kit components).

Plasmid-Delivered Transgenes

  • The TUNR Plasmid-Delivered Transgenes kit allows you to control the expression of a transgene. Simply transfect your cells with one of our four plasmids containing your gene under the control of TUNRs of different strengths (more information about the kit components).

 

Understand the background of the TUNR System - Read this Nature Comm. publication.

Request Quote/More Information  

 

How the TUNR Flexible Gene Editing System Works

Introduction of the TUNR polyA track results in reduced gene expression.

The TUNR Flexible Gene Editing System leverages the CRISPR-Cas9* system for targeted integration of a polyA track at a precise location within the targeted gene.

*Working with ZFNs or TALENs? We also support your genome editing experiments with our TUNR designs and sequences, if you are working with ZFNs or TALENs. Please send your request here.

100 % Control of Your Gene Expression
100 % Control of Your Gene Expression using the TUNR Flexible Gene Editing System

THE SECRET? PolyA Tracks

As recently published in Nature Communications by Sergej Djuranovic’s lab at Washington University in Saint Louis, insertion of tracks of repeated adenosine bases results in decreased mRNA and protein expression.

Shown are wild-type, resulting in normal expression levels, insertion of a short polyA track, resulting in a moderate decrease in expression, and insertion of a long polyA track, resulting in a large decrease in expression. Proteins represented by blue lines.

Adapted from: Arthur, L. L. et al. Rapid generation of hypomorphic mutations. Nat. Commun. 8, 14112 doi: 10.1038/ncomms14112 (2017).

4 reasons to use TUNR instead of RNAi

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:

1) 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.

2) 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.

3) 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.

4) 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.

Request Quote / Request More Information
This site uses the functionality that older versions of Internet Explorer does not support. You should therefore switch or upgrade your browser.