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Last time we introduced CRISPR and its working at a fundamental level. Let's now see how this powerful tool can be manifested to the fullest.
OPERATION OF THE CRISPR/CAS9 SYSTEM AS A GENOME EDITING TOOL
CRISPR-Cas9 is a genome-editing tool that is creating a buzz in the science world. It is faster, cheaper, and more accurate than previous techniques of editing DNA and has a wide range of potential applications.
The CRISPR-Cas9 system consists of two key molecules that introduce a change (mutation?) into the DNA. These are:
· An enzyme? called Cas9. This acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed.
· A piece of RNA? Called guide RNA (gRNA). This consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. This ensures that the Cas9 enzyme cuts at the right point in the genome.
CRISPR Therapeutics During COVID-19 Pandemic
The rapidly advancing CRISPR technology may provide aid during our rapidly evolving times. The recent outbreak of a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic. These pressing times call for an urgent response to develop quick and efficient testing tools and treatment options for coronavirus disease 2019 (COVID-19) patients. Currently available methods for testing are relatively time-consuming with suboptimal accuracy and sensitivity. The two predominant testing methods are molecular testing and serological testing. The US Centers for Disease Control and Prevention (CDC) has developed a real-time RT-PCR assay for molecular testing for the presence of viral RNA to detect COVID-19. However, this assay has a roughly ~30% false negative rate with a turnaround time of several hours to >24 h. Serological testing methods are much more rapid but lack the ability to detect acute respiratory infection since antibodies used to detect infection can take several days or weeks to develop.
CRISPR/Cas9 in cancer
Cancer is a group of diseases characterized by multiple genetic and epigenetic alterations in oncogenes and tumor suppressor genes. Experimentation to manipulate normal and cancerous cell genomes is vital for modeling the disease, as well as for the systematic study of genes involved in the process of initiation, progression, and therapeutic response of cancer.
The fast modeling of genetic events has taken on a significant relevance due to the need to elucidate the importance of genetic alterations present in human tumors, detected by large-scale sequencing of the cancer genome. This is necessary to discern between passenger mutations that are assumed to not directly affect the tumorigenic process and those that directly or indirectly induce mutations that promote the transformation of normal cells into cancer cells by mutating oncogenes (promoting gain of function) and/or inactivation of tumor suppressor genes (promoting the loss of function).
Through the CRISPR/Cas9 technology, it was identified that NANOG and NANOGP8 genes contribute to the high malignant potential of prostate cancer. Knockouts of NANOG and NANOGP8 in human prostate DU145 cells significantly attenuated the malignant potential, including sphere formation, anchorage-independent growth, migration ability, and drug resistance, as compared with that of parental DU145 cells. Cell proliferation was not inhibited in vitro, but in immune-deficient mice, the tumorigenic potential decreased significantly in vivo. To assess the impact of BCR-ABL fusion on the leukemic processes in the Boff-p210 cell line, a hematopoietic cell line that is independent of interleukin (IL)-3 and expresses BCR-ABL p210, CRISPR/Cas9 was used to eliminate expression of the p210 oncoprotein. This resulted in the loss of the ability of the Boff-p210 cell line to grow in the absence of IL-3 and showed a significant increase in apoptosis levels. In immunosuppressed mice, the edited BCR/ABL cells developed smaller tumors compared with those originating from the parental Boff-p210 cells. Furthermore, a single-cell clone of edited BCR/ABL cells with a unique frameshift mutation (averting expression of the p210 oncoprotein) was unable to develop tumors, similar to the results in the Baf/3 parental line.
Authors: Manav, Mudrax, Harshvardhan
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