Gene Editing

Gene-Editing-Spotlight-Cover The rapidly advancing developments in gene editing technology have numerous implications and potential applications for the cell and gene therapy field – from disease modelling through to the potential editing of patient-derived cells to modify/correct disorders.

Gene Edit Detection using Droplet Digital™ PCR

Bio-Rad 


Overview of Droplet Digital™ PCR Technology

Droplet Digital PCR (ddPCR™) enables precise, reliable, and highly sensitive quantification of nucleic acids. The technology is based on water-oil emulsion droplet chemistry which fractionates a DNA sample into about 20,000 droplets within a single well. PCR amplification of the template DNA occurs within the individual droplets via specific primers and fluorescent probes, causing the droplets to fluoresce if the target DNA is present. Each droplet is then read in a droplet reader to determine the number of fluorescent and non-fluorescent droplets. These data are analyzed using Poisson statistics to determine the target DNA template concentration in the original sample with extremely high accuracy and precision.

Droplet Digital PCR for Gene Edit Detection

Genome editing has become a widely used tool and allows for specific modification of almost any cell’s genome. However, the often low (<5%) frequency of gene edits, in particular for primary or induced pluripotent stem cells, hinders simple and reliable detection by gel- or sequencing-based methods, or requires laborious clonal isolation of edited cells. Droplet Digital PCR is a powerful technology for ultrasensitive detection and quantification of NHEJ- and HDR-edited alleles in heterogeneous cell pools. This rapid, cost-effective method is amenable to high throughput and provides a convenient readout for the detection of edited alleles and optimization of editing protocols.


Nucleofector™ Technology for Efficient Transfection of CRISPR, ZFNs and TALENs


Lonza


One common pre-requisite for successful modification of genomic DNA via genome editing tools like ZFNs, TALENs or CRISPR/Cas9 is the efficient co-transfection of involved substrates into the cell type of interest.

Lonza’s non-viral Nucleofector™ Technology is an improved electroporation-based technology which has proven to work as a reliable and sufficient method for delivering the required nucleic acid (e.g., plasmid DNA, mRNA, PCR cassettes, ssODN) or protein (e.g., RNP) substrates into various hard-to-transfect cell lines, primary cells or stem cells, e.g., primary T cells, human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSCs).

Benefit from:

  • Highly efficient transfection of a broad range of cell types, including iPSCs
  • Same transfection conditions for different substrates enabling efficient co-delivery and easy switch between substrate types
  • Proven technology with more than 30 publications on ZFN, TALEN and CRISPR delivery, including high ranking journals

There are different Nucleofection platforms available serving different needs - from lower to higher throughput (e.g. 96-well or 384-well systems for CRIPSR library screens) and from smaller to larger cell numbers.