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"I’ve been working with iTL over the past 5 years in the production of 3 different genetically altered mice. Not only did iTL help in the design of the mice, […]” - Raghu Mirmira, MD, PhD University of Chicago.
CRISPR gene knockout techniques have become highly popular in recent years, as scientists continue to use the technique to research the influence that certain genes have on the mouse genome. Knockout mice offer impressive insight into the inner workings of the human genome due to their genetic similarity to humans. Together with the ease and effectiveness of the CRISPR/Cas9 system, researchers are now able to accurately target single or multiple mouse genes for knockout, opening the door to new studies that could change the future of biomedical research.
Since it’s only been available for about six years, the CRISPR Cas9 gene editing system is still considered new by many standards. CRISPR gene knockout techniques are among the most important achievements of this technology, allowing scientists to accurately target, mark and remove certain genes within a selected genome. Mice are typically used for this purpose and are generated by injection of Cas9 mRNA and single guide RNAs (sgRNAs) into mouse embryos to generate precise knockouts. The manipulation of the target cells occurs via the cell’s homologous recombination machinery. Mice that develop from these embryos are genotyped to determine if they carry the desired knockout, and those that do are bred to confirm germline transmission.
One of the main advantages of CRISPR gene knockout is that scientists now have an easier way of obtaining the models that are ideal for their research. There are various ways to introduce the knockout with gene editing. For instance, the Cas9 system is capable of targeting the mammalian genome either by the injection of a plasmid vector expressing the guide sequence along with a humanized Cas9 endonuclease, or through the co-injection of CRISPR guide RNA. The cleavage of specific genetic coding is repaired by the cell’s own internal mechanisms, leading to insertion or knockout depending on how the experiment is designed. Also, the CRISPR/Cas9 system simplifies the entire process of creating knockout mouse models, and manages to reduce the required time from 1-2 years with conventional methods, to a period of about 6 months.
The CRISPR gene knockout method is certainly not perfect. Since the molecular mechanism exploited by the DNA editing technique is mediated by the cell’s internal instructions for DNA repair, there is the possibility that additional modifications might happen. Deletions, as well as partial and multiple integration of the targeting vector could occur. Even duplication is possible in some cases. Moreover, identifying the desired allele when using the technique directly on embryos is still greatly limited, although scientists are currently working on possible alternatives and solutions to this problem.
Despite any limitations, the CRISPR gene editing technique works extremely well when used on mouse embryos. The generation of simple knockout alleles is one of the main practical capabilities of the system that scientists tend to praise the most. The system has shown great promise when it comes to the creation of specific mutations in laboratory conditions. The sensitivity and accuracy of the system has allowed scientists to create mouse models that mimic human genetic traits and disorders more accurately than before.
Since the development of the CRISPR gene knockout and gene editing technique in 2013, it has already made significant progress. First the technology went from single gene disruption to the ability of targeting multiple genes. Research on its use for achieving point mutations is well established, and today it’s being applied to an increasing extent for the creation of conditional knockout models. For the future, researchers have high hopes that the CRISPR technique will allow them to investigate different areas of physiology much more easily than before. Also, the recent modifications to the Cas9 endonuclease to allow CRISPR to perform tasks that were previously not possible shows that there might be many more options in store, aside from the ease of CRISPR gene knockout techniques.
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