In March 2016, Mark J Osborn et al published in Molecular Therapy a major article for genome editing (doi:10.1038/mt.2015.197), about knock-out of CD3 in human T-cells. The goal is to improve T-cell-based immunotherapies to fight tumours using engineered allogenic T-cells from healthy donors. It is a very good example of how CRISPR-CAS9 can help medecine. And even if you are not very comfortable with CAR T-cells and the treatments of malignancies, I would recommend you read it and especially take a look at figure 2. Indeed, dear friends of genome editing, the authors made a clear and fair comparison of several KO strategies, covering all the main options. Thus, it is not only a major step for anti-tumour treatments but it is also an excellent overview that reveals the best approaches. So, before reading this post any further, you might like to read the article mentioned above. [Read more…]
Stable expression in a cell lines is very useful for numerous projects. For example, it helps in Eukaryotes for optimization of protein productions and it is convenient for recurrent needs and high yields. Stable exogenous expression of tagged protein allows extensive tracking and localisation of the protein in live. Cell lines stably expressing a protein can be used for various screenings such as lost of function assays (CAS9-expressing cells), and reporter-based assays (Gaussian luciferase). Of course, the stable expression could also be shRNA for knock-down.
Stable expression implies insertion into the genome. It used to be random and could cause hits with side effects. Now, targeted insertion in Safe-Harbor sites is now possible. How can you take advantage of this progress?
Mouse ROSA26 and Human AAVS1 safe harbor sites
First of all, what is a safe-harbor site? It is an ideal site in the genome in which we can add a construct without harm and expect consistent level expression. For years, researchers have looked for it. In 1997, Zambrowicz et al initiated the discovery of the ROSA26 site on Mouse chromosome 6. It was shown it is a transcriptionally active region with an open chromatin configuration and transgene insertion has no or minimal effect on global and local gene expression. Remarkably, a ROSA26 inserted transgene is expressed in all tissues.
Similarly, in the human genome there is a safe-harbor site on chromosome 19 (locus PPP1R12C) called AAVS1. It was described more recently by DeKelver, et al. (2010). It has become a remarkable safe harbor site for ESC (Embryonic Stem Cells) and iPSC (induced Pluripotent Stem Cells) because of the robust expression of harboring constructs and the absence of abnormalities or differentiation deficits.
Since absence of visible effect doesn’t mean there is no at all risk of effect, I should mention that an ideal genomic safe-harbor doesn’t exist yet. Nevertheless, AAVS1 in human and ROSA26 are certainly the safer harbor sites today.
Comprehensive Safe-Harbor kit
Today we can easily insert a construction into a targeted site of the genome and so maintain its integrity. The principle is based on the targeted insertion of a Donor construction as illustrated in figure 1.
Each kit includes vectors expressing the system (CRISPR-CAS9) and the following:
- Donor vector in which you clone your ORF of interest
- RFP Donor control to monitor in fluorescence the knock-in
- Primer pairs for the PCR analysis of the genome integration
You can also obtain the Donor vector with your ORF of interest upon request.
The RFP control is highly convenient allowing a direct monitoring of the transgene genome integration (Figure 2).
Comparing with the control (without Donor), we can see the high integration efficiency after only 12 days of puromycin selection.
Comparing CRISPR-CAS9 Safe-Harbor to the classic method
Well… Think easier, faster, more reliable and cleaner!
So, is it a new way to work? As soon as you get the Safe-Harbor kit (with the empty Donor vector), you can use it indefinitely to establish promptly isogenic and polyclonal cell lines expressing all the ORFs you need. And furthermore the cell lines will be more reliable than random integration of lentivirus or plasmid.
Any questions? Please feel free to get in contact by leaving your comments below!
The CRISPR-CAS9 system may well have opened Pandora’s box, but it is also definitely the cornucopia of genome editing.
We can do what we want in the genome: settle a mutation, correct a mutation, insert a fluorescent tag to a protein, add an exogenous gene, delete an endogenous function, suppress a cis-regulatory region, add a reporter…. I might just not have enough imagination!
The main challenge is to define a good strategy, taking in account the specifics of the project and being aware of the corresponding limitations. [Read more…]
For about 5 years now, there has been a renewed interest for mRNA molecules and their numerous applications. Mature mRNAs can be transfected into cells (ex. in mammalian cells) for various purposes (ex. cell reprogramming and iPSCs production, genome editing (such as CRISPR-CAS9), cell line engineering (eg. protein production)) without the need for cloning strategies in expression vectors. In addition, mRNA molecules are now becoming a promising elements in Drug Discovery to deliver genetic information and mRNA-based therapeutics in cells.
Whatever the application used, mRNA production by in vitro transcription (IVT) is tricky and deserves high quality enzymes. In this post, let’s take a closer look at the mScript technology for optimized mRNA productions.
Gaussia Luciferase (GLuc) is a widely accepted gene reporter system used in cell-based assays for various applications (eg. high throughput identification of compounds against viral infections, dose-dependent & pathway-specific inhibitor screenings, functional characterisation of GPCRs, transcriptional activity studies…).
Recently, a modified version (mGLuc) has become available to obtain even more stable and sensitive readout (Figure 1). [Read more…]
RNA-sequencing (RNA-Seq) bottlenecks start with library preparation! Is it possible to prepare a RNA-Seq library in a simple and fast way? And how to avoid the bottlenecks of current RNA-sequencing library preparation?
miRNA are implicated in a number of diseases and cancers. Despite the fact that their different roles in physiological and pathological cell processes are still under investigation, there is already more and more interest in considering them as future key diagnostic and prognostic biomarkers. Indeed, they are conveniently present in blood, and furthermore, their small size makes them relatively robust compared to messenger RNAs. They are well conserved into poor quality samples such as FFPE samples allowing the best hopes when exploring biobanks of tissues.
Accurate and convenient technologies aimed at profiling miRNA expression from such biobanks and blood opens up a promising era in biomarker discovery for personalized healthcare.
But still, we need to find an efficient profiling approach to cover the full miRnome that contains about 2000 Human miRNA… [Read more…]