Gene Editing - A New Frontier

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories
www.abclabs.com

A new technology for “editing” genes has scientists excited about an entirely new method for treating disease. The CRISPR technique, along with other gene-editing technologies (TALENS, zinc finger nucleases) allow for the precise deletion of specific DNA sequences. CRISPR involve a Cas9 protein linked to an RNA strand. The RNA sequence serves to target the specific DNA sequence of interest and once bound the Cas9 enzyme cleaves both strands of the DNA.

Above graphic credit (link) to Scitechweb.com 

Since RNA synthesis is relatively cheap and easy this technology has exploded in the last 2 years. Feng Zhang and Eric Lander at MIT have created CRISPR libraries that target essentially the entire human genome. The promise of the technology is to delete genetic mutations and replace them with the correct genes.  Initial targets involve only a single mutation, e.g. sickle-cell anemia, progressing to diseases involving multiple mutations.

As with any new technology there are calls for caution. Chinese scientists reported attempts to repair defective genes in human embryos. This held up the specter of future “designer babies” enhanced in their embryonic stages. Others warn that this technology could be used to “weaponize” bacteria or viruses. Several prominent scientists have proposed a complete moratorium on gene editing until proper safeguards are in place.

Despite these concerns several biotech start-ups have been founded in the last 2 years including Intellia Therapeutics, Editas Medicine and Crispr Therapeutics. Successful in vitro results have been reported for the repair/modification of clinically important genes in hematopoietic stem cells, fibroblasts and T-cells. A quick internet search yields a number of companies offering their services to utilize CRISPR to perform gene editing in both cell lines and whole animals. Based on the extremely rapid advances in this area, gene-editing using CRISPR and other technologies promises to revolutionize our ability to manipulate the genome and attack genetic diseases.

WES: An Alternative to ELISA

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories
www.abclabs.com

Analysis of protein expression in biological tissues presents numerous challenges. The protein of interest is present in an extremely complex matrix, containing hundreds if not thousands of other proteins, and is typically at very low concentrations. The analysis must be sensitive, precise and most importantly specific. Several methods including Western blot and LC/MS/MS have been utilized, but ELISA is currently the method of choice. The method is specific and sensitive, particularly with the use of electrochemiluminescence detection. However, regardless of the particular format, ELISAs are complicated, labor intensive and time consuming. Coating, blocking, binding and multiple washings present many opportunities for variability resulting in typical CVs of 15-25%.

An alternative to ELISA is an automated Western analysis. ABC Labs has recently acquired the Protein Simple® Wes™ system. This instrument utilizes an automated, quantitative capillary electrophoresis-based Western technique using an antibody against the target protein. Standards, samples and reagents are prepared and loaded into the Wes plate. The plate and a capillary cartridge are loaded into the instrument and the run is started. All subsequent steps are automated. Protein concentrations are extrapolated from a standard curve prepared using the reference standard and a linear fit is used for regression.

Experience in our lab has shown the instrument to be accurate, precise and easy to use. Standard curves were linear with an R² of 0.997 and an LOD of < 1.0 pg/µl.  Analyses of a protein extracted from biological tissue have yielded excellent results: interday/interanalyst CVs averaged <15% with accuracy ranging from 100-104%. Based on our experiences, ABC Laboratories is actively pursuing the replacement of some ELISA methods by those utilizing the WES system.

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