Introduction
Clustered Regularly Interspaced Short Palindromic Repeats, or "CRISPR" (pronounced "crisper"), are the hallmark of a bacterial defense mechanism and the building block of the CRISPR-Cas9 genome editing method. The terms "CRISPR" and "CRISPR-Cas9" are frequently used interchangeably in the field of genome engineering to refer to the various CRISPR-Cas9 and -CPF1 (and other) systems that can be designed to target particular genetic code segments and edit DNA at specific locations, among other uses, like developing new diagnostic tools. In the future, these technologies may allow for the correction of mutations at specific sites in the human genome to treat genetic diseases. Researchers can also use them to alter genes in living cells and creatures permanently. These days, other systems target RNA, such as CRISPR-Cas13s, which offer alternative applications and have special properties used to develop sensitive diagnostic tools like SHERLOCK (SHERLOCK is a development of CRISPR technology, which is used by others to precisely alter genetic code. SHERLOCK can be used as a diagnostic tool to identify the distinct genetic fingerprints that are encoded in almost any DNA or RNA sequence in any pathogen or creature).
How Does CRISPR Work in Nature?
CRISPR is an altered defense mechanism found in bacteria. The bacteria employ the following strategies to repel bacteriophages using the DNA that it store as a defense mechanism:
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A section of the bacterial genome called the CRISPR locus receives DNA from the invading bacteriophage.
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A brief segment of RNA (A, U, G, C) known as a CRISPR RNA (or crRNA) is created during the subsequent bacteriophage invasion by transcription of DNA (A, T, G, C) in the CRISPR locus, which is related to the bacteriophage invader.
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A big protein known as an effector complex, the sequence of which complements the sequence of the bacteriophage DNA, is guided by the binding of crRNAs. Watson-Crick base pairing is used by the effector complex and its crRNA to attach to the bacteriophage DNA that is being invaded.
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The DNA of the bacteriophage invader is broken and saved in the CRISPR locus for future invasions.
How Does CRISPR Operate in a Lab?
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Select the gene that one wishes to cut.
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Create a gRNA that targets a particular PAM sequence close to that area.
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In the target cell, gRNA is expressed with an endonuclease protein like Cpf1 or Cas9.
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Some resultant cells will have loss-of-function mutations in the target gene because the DNA has been cut at that location.
What Are the Uses of CRISPR Technology?
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Treatment of Genetic Disorders: The potential application of CRISPR/Cas9 to cure genetic illnesses resulting from single gene mutations is among its most interesting uses. These illnesses include hemoglobinopathies, Duchenne's muscular dystrophy (DMD), and cystic fibrosis (CF). Although the method has only been proven effective in preclinical models thus far, there is hope that it may soon be able to be used in clinical settings.
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Treatment of HIV (Human Immunodeficiency Virus): Infectious disorders like HIV could be treated with CRISPR/Cas9 in a therapeutic setting. Even though antiretroviral therapy effectively treats HIV, there is presently no cure because the virus has permanently integrated itself into the genome of the host.
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Modifying Somatic Cells in Vivo to Treat Cancer and Other Illnesses: The prospect of modifying patient-derived T-cells and stem/progenitor cells with CRISPR/Cas9 so they can be reintroduced into patients to treat disease has drawn more and more attention. This strategy might be able to solve some of the problems related to effectively delivering gene editing to the appropriate cells. T-cell genome engineering has promise for treating solid tumors, autoimmune disorders, and primary immunological deficiencies. It has previously demonstrated efficacy in treating hematological malignancies.
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CAR-T Cell Therapy: The application of CRISPR is improving cancer immunotherapy. Several labs have employed the method to modify T-cells taken from cancer patients. By inserting particular genes, T-cells can be "programmed" to locate and eliminate tumors inside the patient.
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In Bringing Extinct Species to Life: DNA editing techniques have already brought back the Pyrenean ibex and other extinct species. However, the wooly mammoth presents a far more difficult challenge because DNA deteriorates over thousands of years. Therefore, before being introduced into a viable embryo, the retrieved DNA from the wooly mammoth's hair, skin, and bones must be corrected. CRISPR will make this simpler and more efficient.
What Is the Difference Between CRISPR-CAS9 and Other Genome Editing Tools?
CRISPR-Cas9 is shown to be a more effective and adaptable option when compared to other available genome editing techniques. Unlike other tools, CRISPRs do not require pairing with distinct cleaving enzymes because the CRISPR-Cas9 system can sever DNA strands. Additionally, matching them with specially created "guide" RNA (gRNA) sequences that will direct them to their intended DNA targets is simple. The scientific community has access to tens of thousands of these gRNA sequences. Another feature distinguishing CRISPR-Cas9 from other gene-editing instruments is its ability to target several genes simultaneously.
What Distinguishes CRISPR-cpf1 From CRISPR-cas9?
Numerous key distinctions between CRISPR-Cpf1 and the previously reported Cas9 have crucial research and clinical implications. First, two short RNAs necessary for the cutting activity bind with the DNA-cutting enzyme Cas9 in its native form. It is easier to understand because the Cpf1 system only needs one RNA. Additionally, the Cpf1 enzyme is easier to introduce into cells and tissues since it is smaller than the typical SpCas9.
Perhaps most importantly, Cpf1 breaks DNA differently from Cas9 in the second place. When the Cas9 complex cuts DNA, it does it simultaneously in both strands, leaving behind "blunt ends" frequently changing when they reunite. The two strands' incisions are offset by the Cpf1 complex, leaving brief overhangs on the exposed ends. This will facilitate researchers' correct and fast integration of a DNA fragment through precise insertion assistance.
Third, because Cpf1 cuts distantly from the recognition site, the targeted gene may still be able to be recut, providing several chances for accurate editing to take place, even if the gene becomes altered at the cut site.
Fourth, the Cpf1 system allows more freedom to select target sites. Similar to Cas9, the Cpf1 complex needs to initially bind to a brief sequence called a PAM. Targets for the complex must be selected close to PAM sequences that occur naturally. This is advantageous when targeting the genomes of human beings and malaria parasites.
Conclusion
Researchers may edit the DNA of live organisms selectively by using a technique called CRISPR, which stands for "clustered regularly interspaced short palindromic repeats." The naturally existing genome editing systems found in bacteria were the model for CRISPR, which was modified for use in laboratories. It allows for the quick, easy, and affordable correction of genetic mistakes and the on/off switching of genes in cells and organisms. Functional genomic screens, live imaging of the cellular genome, and the quick creation of animal and cellular models are just a few of the scientific uses for it. Additional possible clinical uses include gene therapy to treat infectious diseases like HIV and the engineering of autologous patient material to treat cancer and other illnesses.
