DNA carries some kind of memory
CRISPR / Cas9 revolutionizes genetic engineering!
Take: CRISPR / Cas9!
Genome modifications with insertion of an intact gene copy into the DNA of the cells are used in a number of diseases, e.g. B. caused by genetic defects, such as those present in severe combined immunodeficiency, are considered to be an efficient therapy. However, there is always the risk of unspecific insertion mutagenesis. The targeted insertion of a gene is much more efficient at a point where a double-strand break had previously occurred and from which it is known with certainty that no essential gene is destroyed and possibly even a degeneration of the cell is induced. A few classes of designer nucleases are now known that can cut DNA sequence-specifically in order to insert the new gene precisely there: the so-called meganucleases, the zinc finger nucleases and the transcription activator-like effector nucleases (TALENs). The disadvantage of these enzymes is that the sequence specificity comes about through the interaction between protein and DNA. In contrast, with CRISPR / Cas9 a short RNA ensures the exact placement of the nuclease. Depending on the target structure, it can be assumed that the efficiency of genome editing via CRISPR / Cas9 is 80% or even higher .
Genome editing in humans
Using the two repair mechanisms, non-homologous end-joining (NHEJ) and homology-directed repair (HDR), various possible applications in human medicine can be derived. NHEJ could e.g. B. can be used to address tumor diseases and infectious diseases. In the world's first clinical study using CRISPR / Cas9, the gene for the programmed death 1 (PD-1) receptor is to be switched off via NHEJ. For this purpose, T cells are isolated from patients, modified with CRISPR / Cas9 and then reinfused into the patient . Four Phase I studies with a corresponding approach in various tumor diseases, including metastatic non-small cell lung cancer, are listed in the clinical studies database at the U.S. National Institutes of Health. All four studies are planned in China. The fact that the principle probably works can be seen from the successful use of the PD-1 inhibitors nivolumab and pembrolizumab in various advanced-stage tumor diseases. Instead of inhibiting the PD-1 receptor with antibodies, Chinese scientists are trying to switch it off at the gene level using CRISPR / Cas9. Whether this is the better approach, however, can be questioned.
In addition, other target genes can in principle be controlled which - if switched off - would improve tumor therapy: Just think of dominant oncogenes such as KRAS or genes that mediate drug resistance. Other ideas to apply CRISPR / Cas9 with NHEJ to tumors target, for example, associated viruses. In cancer cell lines that have been degenerated by human papilloma viruses, genome editing can be used to introduce a double-strand break in the genes for the relevant E6 and E7 proteins. The resulting InDels also eliminate an essential tumorigenic principle.
In general, viral infections - including those that are not associated with tumor development - can of course also be combated with CRISPR / Cas9. In a cell line from a patient with Burkitt's lymphoma, the genome of the Epstein-Barr virus was specifically inactivated against the promoter area with two sgRNAs - and that without any off-target cuts . The various ideas of countering HIV infection with CRISPR / Cas9 are also exciting, with starting points both before and after the integration of the DNA into the host cell genome. If, analogously to the destruction of the PD1 receptor, the co-receptors CXCR4 and CCR5, which HIV needs on the T helper cells to penetrate the host cell, were destroyed via CRISPR / Cas9, HI viruses would no longer be able to infect cells. Somewhat later in the infection cycle, CRISPR / Cas9 can use suitable sgRNAs to attack certain areas of the HIV genome, both the not yet integrated cDNA and the provirus DNA within the host cell genome . This might one day actually make it possible to cure an HIV infection and not just keep it in check.
Another category of dangerous infections is caused by antibiotic-resistant Staphylococcus aureus cells, which could be treated with CRISPR / Cas9 using a special approach. If specific phages are equipped with a Cas9 gene and genes for single guide RNAs that address the bacterial virulence genes, precisely these DNA areas in the bacterial cells are destroyed and Staphylococcus aureus loses its most important weapons .
The examples presented give an idea of how diverse and relatively easy CRISPR / Cas9 can be used to destroy genes by NHEJ. The approach of introducing intact genes into cells using homologous recombination (HDR) is somewhat more difficult, but also very variable, compared to the NHEJ. Here, too, Chinese scientists pioneered the testing of CRISPR / Cas9 on human embryos for the first time . Using appropriate sgRNAs, they addressed various areas in the human β-hemoglobin gene of non-viable human zygotes in order to determine the efficiency of a gene repair using HDR compared to a linkage using NHEJ. In these experiments homologous recombination actually took place in 25% of the cases, which is not all bad, but also not particularly high.
In principle, homologous recombination can cure all possible diseases that are caused by the defect in just one gene, be it cystic fibrosis by repairing the gene for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) or Duchenne muscular dystrophy Repairing the dystrophin gene, or Huntington's disease, by repairing the huntingtin gene. Other diseases such as hemophilia A and B, beta thalassemia, sickle cell anemia or tyrosinemia could also be cured by correcting the respective defective gene. Astonishing successes have already been achieved with these hereditary diseases in mouse models. However, the efficiency of the homologous recombination is still too low and the fear of off-target effects of CRISPR / Cas9 is still too great.
Scientists in Dresden showed how the CRISPR / Cas9 system with HDR could be used to heal tumors. To do this, they integrated 13 different oncogenes into HeLa cells and then tested the extent to which specific sgRNAs address the oncogenes and convert them into harmless wild-type sequences through homologous recombination. In principle, the result was pleasing and also specific. However, unchanged cancer cells still remained, which would not be acceptable in the case of a tumor disease. In addition, off-target cuts were observed in these experiments, the effects of which cannot be foreseen .
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