technology:dna-editing
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technology:dna-editing [2019/12/08 21:19] marcos [CRISPR Cas9] |
technology:dna-editing [2020/04/27 02:20] (current) marcos |
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| - | ===== CRISPR Cas9 ===== | + | ====== Method for DNA Editing ====== |
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| + | ===== Cell Delivery ===== | ||
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| + | ==== Lipid Nanoparticle ==== | ||
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| + | The method provides 90 percent efficacy in gene editing, due to improved delivery, compared to other methods such as AAV. | ||
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| + | "The lipid nanoparticles encapsulate messenger RNA (mRNA) encoding Cas9. Once the contents of the nanoparticles – including the sgRNA – are released into the cell, the cell’s protein-making machinery takes over and creates Cas9 from the mRNA template." (Rees 2019)((Rees 2019 https://www.drugtargetreview.com/article/52485/how-will-crispr-evolve-in-the-future)) | ||
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| + | * Could be made with low-toxicity | ||
| + | * No limit for cargo size | ||
| + | * Immune response much less likely given non-viral nature | ||
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| + | ===== Recombinases ===== | ||
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| + | ==== CRISPR ==== | ||
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| + | "CRISPR processes are still in their infancy, as the current tools are effective at cutting DNA but can result in random repair." (Rees 2019)((Rees 2019 https://www.drugtargetreview.com/article/52485/how-will-crispr-evolve-in-the-future)) Also, despite episomes or alternatively, the use of guide-RNA, offsite non-homologous integration (Deyle and Russell 2009)((Deyle and Russell 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929125)) has been an issue due to its viral background. | ||
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| + | CRISPR is the most popular because of its ease and speed of use. It only takes one day to prepare CRISPR for an experiment. | ||
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| + | === Class2 Cas9 === | ||
| Cas 9 enzyme has a sequence locator called the PAM. When the PAM finds a piece of DNA with its sequence, the enzyme unzips the adjacent DNA to see if it matches the guide-RNA. Matching DNA triggers the Cas 9 enzyme to double-strand-break (DSB) the DNA. A DSB is often not a clean break. Some nucleotides can be lost in the process. | Cas 9 enzyme has a sequence locator called the PAM. When the PAM finds a piece of DNA with its sequence, the enzyme unzips the adjacent DNA to see if it matches the guide-RNA. Matching DNA triggers the Cas 9 enzyme to double-strand-break (DSB) the DNA. A DSB is often not a clean break. Some nucleotides can be lost in the process. | ||
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| Cas9 can also be engineered without DNA-cleaving and purposed for epigenome editing instead. | Cas9 can also be engineered without DNA-cleaving and purposed for epigenome editing instead. | ||
| - | ===== Zinc Finger ===== | ||
| - | ===== TALENS ===== | + | === Class1 Cas3 === |
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| + | "Another development for CRISPR technologies came from researchers at Duke University in the US. The team successfully used Class 1 CRISPR systems for the first time to edit the epigenome of human cells. Conventional CRISPR-Cas9 methods are categorised as Class 2 systems. | ||
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| + | The Class 1 technique makes use of multiple proteins in a process called CRISPR-associated complex for antiviral defence (Cascade). This complex binds with high accuracy to the correct sites. After binding, Cascade utilises a Cas3 protein to target and edit the DNA. They were also able to both activate and repress target gene expression." (Rees 2019)((Rees 2019 https://www.drugtargetreview.com/article/52485/how-will-crispr-evolve-in-the-future)) | ||
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| + | ==== Zinc Finger ==== | ||
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| + | ==== TALENS ==== | ||
| - | ===== Meganucleases ===== | + | ==== Meganucleases ==== |
| - | ===== Site Specific Recombinases SSRs ===== | + | ==== Site Specific Recombinases SSRs ==== |
| "Relying on homology-directed-repair (HDR) for editing risks introducing [[https://en.wikipedia.org/wiki/Indel |indels]] or chromosomal translocations. Even with a precisely targeted nuclease, with HDR, “you’re at the mercy of the cell,” Stoddard observes. For editing without the unpredictability of HDR, he adds, watch for developments in site-specific recombinases (SSRs)." (Tachibana 2019)((Tachibana 2019 https://www.sciencemag.org/features/2019/09/beyond-crispr-what-s-current-and-upcoming-genome-editing)) | "Relying on homology-directed-repair (HDR) for editing risks introducing [[https://en.wikipedia.org/wiki/Indel |indels]] or chromosomal translocations. Even with a precisely targeted nuclease, with HDR, “you’re at the mercy of the cell,” Stoddard observes. For editing without the unpredictability of HDR, he adds, watch for developments in site-specific recombinases (SSRs)." (Tachibana 2019)((Tachibana 2019 https://www.sciencemag.org/features/2019/09/beyond-crispr-what-s-current-and-upcoming-genome-editing)) | ||
| - | However, it can take 100 days to prepare an SSR, whereas CRISPR Cas9 takes one day, so it is speedier for research. | + | However, it can take 100 days to prepare an SSR. |
technology/dna-editing.1575839973.txt.gz · Last modified: 2019/12/08 21:19 by marcos
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