CRISPR-Cas9 System: A Genetic Analysis
Genetic interactions screens are used to detect genetic interactions and, as a result, to provide information into the functions of each gene and their associations with other components in the cell . A genetic interaction is defined as when two genes interact and generate a phenotype that was not expected in light of each of the gene’s effect . In mammalian cells, most of the genetic interaction assays use RNA interference (RNAi) techniques , which is method of silencing a gene based in the degradation of specific messenger RNA (mRNA) molecules. Described initially in plants, RNA interference was also observed in most of the eukaryotic organisms, such as insects, mice, and humans. …show more content…
One of these techniques is the CRISPR or Clustered Regularly Interspaced Short Palindromic Repeats Screens , which is originally used as a mechanism of defense by bacteria and archaea against bacteriophage infection. It was first reported as part of the genome of Escherichia coli and, during the next years, other bacteria and archaea were reported to also present the CRISPR cluster. Cas genes were reported to be conserved in these organisms as well. Basically, CRISPR consists of CRISPR-associated genes (Cas genes) and the CRISPR array, which is a series of direct repeats and spacers that correspond to the nucleic acids of the bacteriophage. In this system, CRISPR arrays are transcribed into an RNA molecule (guide RNA) and then processed into CRISPR RNAs (crRNA) to guide Cas9 nuclease activity against specific portions of the DNA or RNA of the pathogen .
The activity of the guide RNA, which can bind specifically to a sequence of nucleic acids and target the Cas9 nuclease activity, allows the CRISPR-Cas9 technique to be used to edit the genome of bacteria like Streptococcus thermophilus, Escherichia coli, and also of mammalian cells in a specific way. This way, if we use different guide RNAs, we can target specific regions of the genome and, as a result, target the nuclease activity of Cas9 to edit a desired region of the genome