8 ms·
The first thing to realize is that the crispr/cas9 strategy only produces damaged DNA (double stranded breaks, DSBs). It is not supposed to repair the DNA or do
by nonbel 8y ago
The first thing to realize is that the crispr/cas9 strategy only produces damaged DNA (double stranded breaks, DSBs). It is not supposed to repair the DNA or do anything else.
You have five main responses to this:
1) Necrosis/Lysis (cell dies due to malfunction)
2) Apotosis (cell kills itself)
3) Senescence (cell doesn't die but stops dividing)
4) Non-homologous end joining (NHEJ, the free ends of the damaged DNA are just pasted back together by the cells repair machinery)
5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used by the cells repair machinery to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap)
The second thing to realize is that cells containing the targeted DNA sequence will be more likely to have damaged DNA than those without. So once a cell is "mutated" (either because it just existed beforehand, or because it survived the crispr/cas9 treatment), it will have a survival advantage.
The third thing to realize is that often these cells they use can divide multiple times per day, I've seen as high as 12 times a day for activated T-cells. That means in two days a single mutant cell could possibly produce 2^24 = 16,777,216 offspring cells containing the mutation.
Anyway, I think the efficiency of this procedure may have been far overstated. There may be only a very few cells in each experiment that actually get edited, but a few days later when they check, those (already mutated) cells dominate the population due to their resistance to the crispr/cas9 treatment.
Eg, look at extended data fig 1h from this recent paper: https://www.nature.com/articles/s41586-018-0326-5/figures/5 https://www.nature.com/articles/s41586-018-0326-5/figures/5
The more cells that die, the higher percent of "edited" cells later.
- ejstronge 8y ago> 5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap) I wanted to elide the details, but this is what I was referring to when I discussed CRISPR applications that try to change a nucleotide sequence. This of course leaves discussions of base-editing aside[1] 1. http://www.sciencemag.org/news/2017/10/novel-crispr-derived-base-editors-surgically-alter-dna-or-rna-offering-new-ways-fix http://www.sciencemag.org/news/2017/10/novel-crispr-derived-...
- nonbel 8y agoYes, you mentioned the two possibilities that everyone likes to focus on, but not the others which (I suspect) are much more common. EDIT: I really mean that I think in some of these experiments, where they only try to cause NHEJ, they just already had mutants in the population and selected for them. Its possible not a single cell was successfully edited but you still have so many cells with indels at the targeted sequence afterward.
- azeotropic 8y agoIndel mutation rates aren't high enough, and the number of cells screened for mutations is too low for this explanation to suffice.
- nonbel 8y agoDo you have a paper in mind? From what I've seen 0.01% - 10% of controls have indels at any given site (obviously this depends on treatment, cell type, etc). You could call that noise of course, at the very least they can't detect rates below those using current methods. I'll go find some refs on it if you ask.
- azeotropic 8y agoYou must be misunderstanding what you've read. There's no way cells survive with indels at 10% of their base positions, that's not possible.
- nonbel 8y agoThats not what I said. I said some significant percent of cells (I remember as high as 10% from one paper, but usually its closer to 0.1%...) seem to have indels at any given locus. Either that or the methods used to measure this are too noisy to detect mutants that exist at those rates.
- 8y ago
- azeotropic 8y agoExtended data figure 1h is testing the optimum pulse settings for the electroporation machine. There is a well-known trade-off between efficiency and viability (too small a pulse and no DNA is transferred into cells, too strong a pulse and the cells are destroyed). The efficiency increase at lower viability cannot be attributed to pre-existing mutants, because they're assaying for the presence of a GFP transgene inserted by homology-directed repair, not indels in general.
- nonbel 8y ago>"The efficiency increase at lower viability cannot be attributed to pre-existing mutants, because they're assaying for the presence of a GFP transgene inserted by homology-directed repair, not indels in general." Yes, for the HDR case it is usually extremely unlikely (barring contamination) that the mutant already existed. If you reread my other post I specifically refer to the NHEJ case when discussing pre-existing mutants. However, a single T-cell could (T-cells can divide up to 12 times a day[1]) expand to as many as 2^48 = 2.81475e+14 daughter cells in the 4 days of that study (see panel A of the same figure). They started with only 1e6 cells in the culture. Therefore these results could be explained by editing a single cell that then proliferates to make up the 30% of the population (300k cells) they see. [1] http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015423 http://journals.plos.org/plosone/article?id=10.1371/journal....
- azeotropic 8y agoTry reading the methods section of your T-cell paper. After electroporation, they cultured at constant cell density, which only required adding media every 2-3 days. So, on average, only one cell division happened before measurement, unless you want to claim GFP stimulates T-cells to hyperproliferate. My intuition is that cells with the transgene will actually be slower than their competitors, since they have to waste energy to make GFP. The PLoS One paper you've linked is about in-vivo hyperproliferation of T-cells in response to an ongoing infection, not about in-vitro primary culture in minimal media.
- 8y ago