Ledford, H. (2017, March 07). CRISPR: gene editing is just the beginning. Retrieved July 05, 2017, from http://www.nature.com/news/crispr-gene-editing-is-just-the-beginning-1.19510
CRISPR has signaled a new age for molecular biologists, and non-profits like Addgene are on top of it all. Addgene has become the place where scientists deposit their tools while also turning to them in search of molecular tools. CRISPR now resembles something novel for researchers all around the world: Specificity – the ability to do exactly what they want and just what they want. The CRISPR tool can be used in many ways to suit the needs of the researcher. A common technique being used it to observe the effect of a change in the genetic code (particular genes) on the cell. This allows for a deeper understanding of metabolic pathways and effects of proteins and genes. The CRISPR associated protein 9 (CRISPR Cas9) is the enzyme which is in widespread use in laboratories.
Researchers from Stanford University modified the Cas9 to just inhibit the gene, and not cutting the gene (as it would normally). The brilliancy of CRISPR is the fact that it itself has been modified and we can attach molecules that affect the epigenetics of the cell, that can be induced by specific stimuli and we can attach molecules that can even stimulate the expression of a particular gene. The ability of this tool to modify the epigenome with high specificity has massive implications as the epigenome has been linked to issues like cancer and neurological disorders.
Lastly, CRISPR has had a major positive impact on the economical side of research by reducing both the time taken and cost of experiments considerably. For instance, CRISPR has enabled researchers to do what previously took a year in just a month!
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Larson, C. & Schaffer A. (2016, October 04). New Tools for Editing the Genome Could Radically Change the Study of Human Diseases. Retrieved July 05, 2017, from https://www.technologyreview.com/s/526511/genome-editing/
Genetic editing using the CRISPR-Cas method is revolutionizing the world and has transformed the scenario of many labs throughout the world. It now provides a cheap and highly precise method of gene editing. CRISPR is a tool born from the collaboration between scientists from varied backgrounds pursuing a common goal. Recent research in China has proven the possibility of genetically edited macaques and this is a stepping stone towards “designer babies”.
The introduction of ethical dilemmas due to the advent of this technology is an inevitable repercussion of any powerful tool. However, the success of the aforementioned experiment seeks to showcase the importance of primate models in research. Primate models have also been useful in understanding genetic diseases and mutations and CRISPR now allows researchers to identify these mutations and find gene therapies to it.
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Kiefer, J., Yin, H. H., Que, Q. Q., & Mousses, S. (2009). High-throughput siRNA screening as a method of perturbation of biological systems and identification of targeted pathways coupled with compound screening. Retrieved July 05, 2017, from https://www.ncbi.nlm.nih.gov/pubmed/19597791
There are two ways to screen cells containing the CRISPR Cas9 nucleases that knockout specific genes: pooled format and arrayed format. In arrayed screening, the cells and other molecules are arranged in multi well plates (96 wells, 384 wells, or 1536 wells) and the cells that are placed in each of the wells are transfected or virally transduced individually (on a well to well basis). The wells are imaged and then the readout is based upon measurements of those images based on different parameters.
In the pooled format, the reagents are synthesized in a pool and are then virally transduced into the cell. Viral transduction ensures that generally only one change to the cell occurs and the integration of the virus can be read through polymerase chain reactions and next-generation screening. The readout of the experiment is based on the abundance of the integrated transgene between samples (in positive selection groups, control groups, and negative selection groups).
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Phillips, T. (2016, October 14). What Is siRNA and How Is It Used to Attack Protein Cells in the Body? Retrieved July 05, 2017, from https://www.thebalance.com/what-is-sirna-and-how-is-it-used-375598
RNA interference (RNAi) is the usage of small interfering RNA (siRNA) that bind to the messenger RNA (mRNA) for specific proteins and cause their degradation hence inhibiting protein synthesis. The siRNA uses a combination of endonuclease and exonuclease enzymes to bring about the degradation of the mRNA.
The problem with siRNA is that it can be rejected by the cell and may trigger immune responses. Also, RNAi is known to give false hits and can have an impact on multiple metabolic pathways/mRNAs and cause an unspecific, unwanted, and unknown knockout. However, this method is still very useful in stabilizing serums, enhancing the activity of cells and is also being used in designing therapies for genetic diseases.
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Tan, J., & Martin, S. E. (2016, December 28). Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening. Retrieved July 05, 2017, from http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0168968
The research conducted by Tan, J. and Martin, S. E. proves that the viral transfection based CRISPR Cas9 method can be used in the arrayed format by comparing the results of various different techniques and finding conclusive evidence that the results obtained from the arrayed format screening of the cells is similar to that obtained from using the pooled format and RNAi method. They tested their results based on the assay for aberrant DNA replication.
Their findings show that the majority of the transfected cells showed a change in phenotype within 72 hours and also that high throughput gene editing and screening of the cells produced data (known DNA replication regulators ) that was comparable to that obtained from siRNA gene knockdown. Lastly, their findings also show that CRISPR screening gave less false hits, i.e. it was more accurate than siRNA screens performed in parallel.