CRISPR : A tool for genome editing

By Edgardo Lopez

            Streptococcus pyogenes is the bacteria that were discover that has the capacity to knockout exogenous genes when is infected by a virus through non-homologous end joining (NHEJ). This adaptive mechanism is call CRISPR (Clustered Regularly Interspaced Palindromic Repeats) and in recent years this tool is been use for sequence-specific editing of mammalian genome. This knockout tool has been very efficient in NHEJ-induced indel mutations but in point mutations or sequence fragments directed by a homologous template is bean poorly efficient. In this article I’m discussing the new approaches to this tool and the optimization in the technique for a better genome editing. CRISPR is a great technique to achive genome editing for both biomedical research and clinical application s like possible specific knockout of genes that are related to a certain disease. Since this technique is practically new it have some poor performance in the specificity of editing. Chen Yu and his colaborators had found four small compounds that can enhance or inhibit CRISPR. The two enhancers are called L755507 and Brefeldin A. L755507 is a beta 3-adrenergic receptor agonist that increased the efficiency of gene insertion and Brefeldin A is an inhibitor of intracellular protein transport from the Endoplasmic Reticulum to the Golgi apparatus. These two enhancers are now known as molecules that significantly improve the insertion fo new genetic information into the cell.  The two inhibitors are called azidothymidine(AZT) and Trifluridine(TFT) and both of them are thymidine analogs. AZT is also use as an anti-HIV drug inhibiting reverse transcriptase and TFT as an anti-herpes virus drug that blocks DNA replication. These two inhibitor of insertion are now well known in this process, but they also enhance deletion of DNA theorizing that the two processes are competitive actions in the cell Through the process of validation this group of scientist perform cytotoxicity analysis, plasmid transfer to different mammalian cells, chemical screening to see activity, validation of the enhancing and repressing compounds using flow cytometry analysis and analysis of gene insertion.

The development of high-throughput efficient compounds form CRISPR genome editing has result in a better way to interact with NHEJ or homology-direct DNA repair pathways. The identification of the chemicals doesn’t exhibit high toxicity and works great in different types of cells. Upgrading genome-editing tools can lead to better ways of insertion, replacement of even removal of DNA in human cells. By mentioning those approaches I’m looking forward in the future  for new drugs that can be more personalize and more efficient and specific.

Reference:

Chen Yu, et. al.  2015.  Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells. Cell Stem Cell 16, 142-147 http://dx.doi.org/10.1016/j.stem.2015.01.003

WHOLE GENOME SEQUENCING MAY IMPROVE PATIENT MANAGEMENT (SCIENTIFIC)

 By CESAR PEREZ

Different diseases have different phenotypes, but there are some diseases with remarkable differences inside the same problem. Consequently, there is a difficulty in detection and treatment. Bainbridge and his team presents a study were they show advantages on the using of whole genome sequencing in the search of variants inside people's genome in order to detect high frequency variants for discriminating heterogeneous diseases from another kind of problems or variants that are benign.

For the study described, the scientists focused on Dopa (3,4-dihydroxyphenylalanine)–responsive dystonia (DRD), a disease with Mendelian inheritance. DRD is a clinically and genetically heterogeneous disorder which makes it ideal for searching the coverage of whole genome sequencing as a clinical tool. However, there is a difficulty on the diagnosis of DRD which is symptoms variation along the day, it is very common to find reduced dystonia by awakening and increasing distonia during midday. Traditionally, differential diagnosis for DRD was applied including  early parkinsonism and cerebral palsy. On the other hand, clinical diagnosis is focused in a neurological scope, inheritance, age, presence of certain metabolites, and the response to L-dopa treatment.

Scientist realized a whole genome sequence of twins diagnosed with DRD, its unaffected older brothers, unaffected parents, and cousins. Then, all the genetic data was analyzed in order to found single nucleotide polymorphisms (SNPs). In addition, affected twins were treated with L-dopa and were monitored. Among the results obtained, a set of putative nucleotides were found which were probably related to DRD. In order to validate the variant sites, a set of primers was developed, then PCR was realized. Actually, designed primers were able to detect the mutation in affected twins, and parents or relatives. Finally, Twins were treated, and after 4 months of treatment presented improvements in focusing in school and  athletic activities.

This work was important due to the power of whole genome sequencing could help in the diagnosis and treatment of DRD, plus another highly heterogeneous symptoms diseases. Sequencing methods not only could be used to diagnosis, but also to found risk on inheritance of different disorders. In addition,  the analysis of genomes presents more and more advantages like less time consumption, analysis of the whole picture of a disorder, or the discovery of new sites related to diseases. Nevertheless, this technique must face challenges yet: information among the patients,  unknowing variants and functions, undercovering of the relationship between variants alleles and phenotypes, or the develop of practical medical databases for the variants' information.

References:

Bainbridge, M. N., Wiszniewski, W., Murdock, D. R., Friedman, J., Gonzaga-Jauregui, C., Newsham, I., ... & Gibbs, R. A. (2011). Whole-genome sequencing for optimized patient management. Science translational medicine, 3(87), 87re3-87re3.