User:KohakuHearts/sandbox

Conditional Gene Knockout is my choice of topic as it seems a very interesting topic to explore the methods, that geneticists, are using to knockout genes from designated areas. As I am also reading a book about IGM's, it is also an interesting expansion to knowledge I am gaining by reading the book.

CRISPR Method
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a relatively-new technology, adapted from the adaptive immune system-based response used by both bacteria and archea, which can be used for conditional gene knockout due to its specificity in sequence targeting. In terms of eukaryotic organisms, a target vector with pre-made complementary DNA sequences must be created and microinjected into the desired organism's initial developmental phases (E.x - Cytoplasmic or Pronucleic Zygotes) and specific tissue. Once injected into the harvested zygotes, the Cas9 nucleases require two complementary sgRNA (Signal Guide RNA) in order to target and attach to a desired cleavage site. A double-stranded break is created in the complementary sequence of the targetted allele, using paired(Two types)of nuclease (Cas9) domains which will insert a pre-made sequence containing two loxP site and the exon to be floxed. The single DNA template also consists of two arms with homologous genomic sequence immediately upstream (5') and downstream (3') of the targeted exon which will be deleted by two loxP sites. Using Cre recombinase technology with the two implanted loxP sites, a frame-shift mutation occurs which is followed by a nonsense-mediated mRNA decay, and the knockout of a specific gene within the cell will occur and will be known as the Floxed gene/exon. In order to verify the success of conditional gene knockout, primers can be used to screen for the deletion products depending on the organism. Other methods could be inserting a marker within the DNA template sequence or inserting a GFP-reporter gene in order to detect deletion. Cre-mediated recombination may leave a DNA byproduct, thus primers can be used once again to detect their presence.

CRISPR has the capability to trangenically alter the germline of organisms using conditional knockout. The CRISPR technique is faster and more specific than altering ES(Embryonic Stem) cells, when it is successful. In 2014, in the study conducted by A. Lee et al., there was only a 8%( 1 out of 12 mice) success rate when detecting the mutant allele in progeny of CRISPR mutated mice. It is being investigated as a more efficient and less time consuming method compared to the current implantation of designated genes within ES Cells, as well as a possible mechanism for humans as the ES Cell method produces chimeric intermediate species. Thus, although CRISPR is a very plausible method of developing conditional gene knockout, it will require time to refine the technique in order to be used on humans for the same purpose.