When it comes to the experimental aspect of using CRISPR, knockdown genes are one of the few that truly captivate the attention of scientists all around the world. While knockouts and knock-in genes can offer tremendous insight into the inner workings of the mammalian genome, knockdowns are emerging as a valuable source of research. With CRISPR, obtaining knockdown genes can be far easier than with other techniques, even if they have been used for a long time. Moreover, the future of CRISPR is bright, as a growing number of scientists from all around the world are adopting the technique and adapting it to their research.
So far, when using CRISPR, knockdown techniques aren’t the bulk of the research that most scientists focus on. Gene knockdown is still considered an experimental technique, that however, could prove useful for future practical applications. A knockdown is essentially a gene that had its expression reduced, instead of being stopped altogether (as in the case of a knockout). Knockdown genes can be obtained either through genetic manipulation, through CRISPR, or with reagents such as RNA oligonucleotide or a short DNA.
Aside from the use of CRISPR, knockdown genes can also be obtained with the help of oligonucleotide that temporarily bind to a gene, leading to a temporary or “transient” knockdown. Transient knockdown methods are commonly used in developmental biology, and are frequently referred to as “reverse genetics” techniques. They involve a number of possible options, such as gene binding, the blocking of mRNA translation or the use of siRNA (small interfering RNA).
When it comes to the use of CRISPR, knockdown is more easily obtained than with any other technique, as many experts can admit. The new system was discovered with the help of prokaryotes, although researchers can now use CRISPR for genome editing, and specifically knockdown gene creation, featuring eukaryotic cells as well. The technique for obtaining knockdowns with CriSPR entails the use of proteins known as CRISPR-associated genes, which can be programmed to insert exogenous DNA fragments into a CRISPR repeat locus. This method allows for the production of small RNAs that can serve as a template for Cas proteins to silence that same sequence in a cell. CRISPR is commonly used by many researchers for obtaining knockdowns in various mouse models, for the purpose of fine-tuning their genetic research. The main goal is usually to find the precise mechanism through which the silenced genes work, and what happens when their expression is diminished or stopped.
Aside from CRISPR, knockdown genes can also be obtained through various other means. TALENs and RNA interference are two of the best known methods, and they also enjoy nearly as much popularity as the CRISPR technique. RNA interference uses mRNA degradation to obtain silencing effects in specific genes. This is primarily a research tool that allows for easy screening, and makes it possible for researchers to determine what area of genetic research to focus on next. TALENs is another common method of obtaining knockdowns, which however, focuses on genome manipulation with the help of transcription activator-like effector nucleases (TALENs).
When it comes to knockdowns, commercialization is not yet underway in full force, since the method is only in its infancy. Presently, researchers have only just scratched the surface on what is possible with the technique. Knockdown animals such as rats and mice are most commonly used for reverse genetics purposes, and there are several companies that make knockdown mouse models available commercially. With the help of CRISPR, knockdown genes are easier to obtain than ever before, and the technique shows great promise for future study.
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