CRISPR-CAS9 is a powerful technology for gene editing. It allows targeted modifications into the genome of the cell lines, IPSCs and ESCs. Despite the principle being pretty simple, in practice its use requires some expertise, and it can even turn into a time consuming adventure… That’s why the best way to benefit from it is most certainly through affordable services.
Many research labs would like to acquire and implement the CRISPR-CAS9 technology for their gene editing projects. Indeed, it’s a powerful system based on a simple principle: an endonuclease, the CAS9, driven onto a target site by a short guide RNA. There are so many strategies with benefits and drawbacks that is quite challenging to figure how to start out. Newcomers may be pushed into necessarily becoming experts before finding an efficient way to success. But what if you could use a complete and simple kit to facilitate your projects?
Vector-free CRISPR-CAS9 gene editing to accelerate therapeutic applications
A few years ago, Ayal Hendel et al (doi:10.1038/nbt.3290) published results revealing that chemical alterations to sgRNA enhance gene editing in primary cells. To demonstrate this, Matthew H Porteus’s team chose the targeted genes CCR5, HBB and IL2RG respectively involved in anti-HIV clinical trials, cell anaemia and thalassemia, and severe combined immunodeficiency. More recently, the same team tested several modified CAS9 mRNA. You can find the practical results on this poster introduced at the ASGC. [Read more…]
Shen et al (Nature Methods, 2017) explore and identify synthetic interactions among 73 cancer-associated genes. To perform their loss of function screen they combined CAS9-expressing cell lines with a sgRNA library of high titer lentiviral particles. Most of these gene interactions were subsequently validated by drug treatment. [Read more…]
Since the discovery of reprogramming factors in 2006 and the boom of CRISPR gene editing strategies, induced pluripotent stem cells (iPSC) have emerged as new cellular models. The development of 3D cell culture technologies has also contributed to the generation of induced Pluripotent Stem Cell (iPSC) derived cells, with unique applications from patient-specific drug responses testing, to regenerative medicine. I would like to introduce in this post a selection of reagents in this domain, a combination of both routine and innovative quality reagents, that I consider as bringing something extra to your stem cell research projects.
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…]
The introduction of transgenes into stem cells has shown to be a valuable experimental technique for studying stem cell biology. Transfecting stem cells without inhibiting cell viability and cell growth has shown to be difficult. DNA-In® Stem Transfection Reagent offers a simple, robust and reproducible method for delivering DNA into a wide range of stem cells, including neural stem cells. Formulated and optimized specifically for embryonic and adult stem cells, DNA-In® Stem is a new-generation transfection reagent that enables high efficiency transfection while maintaining maximum cell viability and cell growth.
In this post, I invite you to discover the benefits of using DNA-In® Stem Transfection Reagent vs. other reagents. A lot of pictures and graphs rather than long descriptions! Last but not least, DNA-In® Stem Transfection Reagent is less expensive compared to Lipofectamine reagents… [Read more…]
CRISPR-Cas9 is a popular method that brings researchers endless experimental strategies to create their own research-based cellular models. In this post we’ll review a new transfection reagent especially engineered to maximize Cas9 vectors deliveries inside cells with low cellular toxicity.
Many labs have adopted the CRISPR genome editing technology to make knock-out and knock-in cell lines.
This technology produces first a targeted break in genomic DNA, which can then be exploited to produce cell lines with genes knocked out or where a donor vector has been used to introduce new genetic elements (point mutants, fluorescent tags, antibiotic resistance cassettes, etc.). Essentially any desired modification to the cells genome can be made. In setting up these genome editing projects there are many choices to be made including vector for the Cas9 protein and for the sgRNAs. Perhaps the most difficult choice, however, can be which cell line to use. Even the most affordable stable genome editing cell line development services can come with a significant cost, so choosing the right cell line at the beginning is crucial. Here we explain some of the choices researchers have in setting up their CRISPR genome editing projects and give our advice for cell line selection.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) and Transcription Activator-Like Effector Nuclease (TALEN) are endonuclease based technologies aimed at developing targeted genome editing technologies.
CRISPR and TALEN provide Scientists with unique discovery tools for pathophysiology or genotype-phenotype studies by creating cellular models with gene knock-out, knock-in or tagging, promoter swapping, nucleotide substitution, protein truncation, reading frame disruption, modification of regulation by miRNA, genetic defect corrections…But, which one is the best for your application?