CRISPR-Cas9 has been wildly used to generate gene knockout models through inducing indels causing frame-shift. Researchers can use CRISPR to generate knockout cells or animals by co-expressing an endonuclease like Cas9 or Cpf1 and a sgRNA specific to the gene to be targeted. Generating a CRISPR knockout is easier than ever with advanced and highly simplified techniques like CRISPR-Cas9. With CRISPR-Cas9, researchers are now able to accurately knockout single or multiple genes, opening the door to a myriad of different possibilities for study, that could change the face of medical and genetic research. CRISPR-Cas9 knockout has emerged as a powerful method to interrogate gene function.
CRISPR-Cas9 knockout techniques are among the most important achievements of this technology. Scientists have used the technique countless times 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 and the effectiveness of the CRISPR technique. 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.
CRISPR is first transcribed to precursor CRISPR RNAs (pre-crRNAs) and then processed into crRNAs, which assemble with cas protein to form a complex that is able to trigger and cleave target DNA sequences. CRISPR-cas9 system is composed of cas9 nuclease and a chimeric single-guide RNA (sgRNA) engineered from crRNA and a trans-activating crRNA (tracrRNA); crRNA is responsible for recognizing and binding the sequences next to protospacer-adjacent motif (PAM) on the target DNA, whereas tracrRNA is essential to maintain the cas9 nuclease activity. sgRNAs direct Cas9 to the complementary target sites and at this site Cas9 nuclease cuts the double-strand DNA to generate double strand DNA breaks (DSBs).
Repair of these DSBs can through the pathway of non-homologous end-joining (NHEJ), which is able to introduce variable sizes of insertions or indels. If the indels is not a multiple of three nucleotides shift, it is able to shift the reading frame and introduce premature termination codons (PTCs), which may result in mRNA degradation by nonsense-mediated decay (NMD), thereby making the gene loss function. Eventually, the expressions of target genes can be interrupted by the frame shift occurring in the coding regions. Over the past few years, CRISPR-Cas9 system has been proved to be a simple way to generate loss-of-function (LOF) mutations in the genome of many organisms, including mammals.
One of the main advantages of CRISPR-Cas9 gene knockout is that scientists aren't forced to only consider one option when it comes to gene editing. For instance, the CRISPR-Cas9 system is capable of targeting the mammalian genome in two distinct ways: by the injection of the plasmoid vector expressing the guide sequence along with a humanized Cas9 endonuclease, or through the co-injection of CRISPR guide RNA. 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 the help of conventional methods, to a period of about 2 months.
Because the CRISPR-Cas9 knockout 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 NHEJ repair technique-the CRISPR knockout method is considered simpler and more elegant than other knockouts. Since one can design his own sgRNA, one 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. HDR requires a repair template and displays reduced efficiency compared to NHEJ knockout.
Although it has many advantages, the CRISPR-Cas9 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 no way to control any additional modifications that might ensue. Deletions, as well as partial and multiple integration instances of the targeting vector could come up. Even duplication could occur 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 the problem.
Despite any limitations, the CRISPR gene editing technique works extremely well when used on mouse embryos. The generation of simple alleles in knockout point mutations is one of the main practical capabilities of the system. The sensitivity and accuracy of the system has allowed scientists to create mouse models that mimic human genetic traits and disorders more accurately than ever before. The CRISPR technique will allow researchers to investigate different areas of physiology, such as reproductive mechanisms, much more easily than before. Also, modifications to the Cas9 nuclease to allow CRISPR to perform tasks that were previously impossible shows that there might be many more possibilities in store, aside from the perfection of CRISPR gene knockout techniques.
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