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--- technology:dna-editing [2019/12/07 06:42]
marcos created
+++ technology:dna-editing [2020/04/27 02:20] (current)
marcos
@@ Line 1: / Line 1: @@
-===== CRISPR Cas9 =====
+====== Method for DNA Editing ======
+===== Cell Delivery =====
+==== Lipid Nanoparticle ====
+The method provides 90 percent efficacy in gene editing, due to improved delivery, compared to other methods such as AAV.
+"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))
+  * Could be made with low-toxicity
+  * No limit for cargo size
+  * Immune response much less likely given non-viral nature
+===== Recombinases =====
+==== CRISPR ====
+"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.
+CRISPR is the most popular because of its ease and speed of use.  It only takes one day to prepare CRISPR for an experiment.
+=== 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.
@@ Line 6: / Line 28: @@
 The host DNA repair mechanisms come about to repair the DSB, and if a homologous DNA strand is around, it will be used as a template for repair.  The homologous DNA can be provided as part of the technological payload, for the purpose of altering the genome.
-===== Zinc Finger =====
+Cas9 can also be engineered without DNA-cleaving and purposed for epigenome editing instead.
+=== Class1 Cas3 ===
+"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.
+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))
+==== Zinc Finger ====
+==== TALENS ====
+==== Meganucleases ====
-===== TALENS =====
+==== Site Specific Recombinases SSRs ====
-===== Meganucleases =====
+"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))
-===== SSRs =====
+However, it can take 100 days to prepare an SSR.

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