Generating a CRISPR Knockout – Important Thoughts on the CRISPR/Cas Technique
Generating a CRISPR knockout is easier than ever with advanced and highly simplified techniques like CRISPR/Cas9. The CRISPR Cas system has been in use for years, and it’s already helped researchers achieve some important breakthroughs in the field of medicine and genetic engineering. With the help of CRISPR knockouts, scientists are confident that they can find the genes and gene functions responsible for a number of diseases and important protective functions, so that genetic therapies can be develop to counteract problems that were believed to be insurmountable.
What Are Knockouts and Why Are They Needed?
There is a lot to consider about knockouts and their role in molecular biology and medicine. Suffice it to say that a knockout is the deletion of a specific, target gene that fulfills a certain function. A CRISPR knockout is simply a knockout gene obtained through the use of methods such as the CRISPR Cas system. The need for knockouts has to do with the study of particular gene functions and how they impact the body. By creating knockout mice, researchers can understand how a certain gene works, and what functions are impaired when the gene is inactivated.
The CRISPR/Cas Technique
Before starting with your preferred method to create a CRISPR knockout, it’s important to have a clear overview of how the process will unfold. The CRISPR Cas9 method is a simplified technique that can easily be used to create gene knockouts at specified, target sequences. CRISPR uses a guide RNA, or gRNA, which instructs the Cas9 protein to mark the target sequences that have to be eliminated and create breakages that facilitate this process. Your goals will, therefore, be to design your guide RNA, synthesize it, and deliver it, along with the Cas9 protein, to your target cells. Once the process is completed, detecting indels and finding your gene knockouts is all that’s left to be done.
Generating an Insertion/Deletion Knockout with CRISPR
Because it uses the cell’s natural processes for repairing breaks caused by the Cas9 protein – methods such as homologous directed repair (HDR), or the more active non-homologous end joining (NHEJ) repair technique – the CRISPR knockout method is considered simpler and more elegant than other knockouts. Since you can design your own gRNA, you can decide precisely which sequence the Cas9 protein will target. The protein will then latch itself onto the target sequence and create a double-strand break. Once this happens, the cell’s imperfect repair mechanism will allow for the easy creation of cell lines that feature indel knockouts.
Planning Your CRISPR Experiment
Before starting to even consider a CRISPR knockout, you have to plan and see what you actually have to do. One of the first requirements is to determine whether or not to use a specific cell line. Which cell line are you using? Are you considering primary or post-mitotic cell lines? The latter will likely present you with better results when you use viral vectors, although the process can be more time-consuming. Additional factors to take into account also include deciding on which type of Cas9 system to use. AAV is good for a maximum cargo size of about 4.5 kb, while a smaller Cas9 might be more appropriate if you need additional control elements. Finally, it’s also important to read up on SpCas9, if you want to make sure you can reduce unwanted, off-target mutations.
The Benefits of Knockout Mouse Models
Knockout mice are some of the most common laboratory animals used for genetic research. Mouse models are easy to develop and can offer many possible genetic enhancements that are relevant to the field of human genetic study. This fact is partly possible because of the fact that mice are genetically similar to humans, but also because humanized mouse models are easy to develop. Mice developed with the CRISPR knockout technique are available for purchase at relatively accessible prices, and you can often get additional benefits such as several genetically independent cell lines, relevant genetic backgrounds and short development time.