6 ms·

Pretty cool visualization. Single molecule work like that is challenging to do properly - but when done properly gives a kind of visual proof to a hypothesis t

Pretty cool visualization. Single molecule work like that is challenging to do properly - but when done properly gives a kind of visual proof to a hypothesis that is hard to controvert. Wild-type Cas9 cuts genomic DNA, and when it does, the strand can go flying - with repair a possibility rather than an inevitability. Cas9 (and similar proteins) have 2 basic functions: 1) to bind to specific DNA (by means of complementing a specifically-bound RNA) 2) to cut the DNA that is has bound. Only one of these functions is actually desirable or unique to Cas9 - that of binding to a sequence-specific part of DNA. The 'cutting' function is how the CRISPR system naturally destroys foreign DNA in Cas9's normal environment, but is generally not desirable in these synthetic biology or therapeutic applications. For genetic engineering purposes we really have better tools than Cas9 to cut, modify, or otherwise affect DNA - but Cas9 is clearly a fantastic tool to localize to particular sequences. That this paper so clearly demonstrates the terrifying damage that cleavage can do to genomic DNA only reinforces that Cas9 is a useful scaffold upon which to engineer further tools, but in its wild-type form is kind of scary. Recent papers from the Liu lab at Harvard [1] (among many others) show how you can edit DNA using a modified Cas9 that has been modified to not have that second cutting function, while retaining the ability to bind to a specific DNA sequence. The synthetic "BaseEditor" protein has been further modified to have more complicated DNA modification capabilities, rather than whole-sale slicing of DNA [2]. Though this designed protein with Cas9 at its core is complicated, it should not cleave and let dangle genomic DNA as we see it here visualized. I'd be curious to see how the BaseEditors behave under a similar visualization. [1] Kim et al. 2017: https://www.nature.com/articles/nbt.3803 https://www.nature.com/articles/nbt.3803 [2] BaseEditor v4: https://serotiny.bio/notes/proteins/sabe4gam/ https://serotiny.bio/notes/proteins/sabe4gam/

nonbel 9y ago
>"Wild-type Cas9 cuts genomic DNA, and when it does, the strand can go flying - with repair a possibility rather than an inevitability." "Possibility" is too strong a term. Afaict, the best you can say about repair is that it is a "less than common occurrence," and even that may be too much. >"Recent papers from the Liu lab at Harvard [1] (among many others) show how you can edit DNA using a modified Cas9 that has been modified to not have that second cutting function, while retaining the ability to bind to a specific DNA sequence." Sorry but I could not find any comment/data about toxicity in this paper. This is basic info everyone wants to know: What percent of cells exposed to this stuff die? Maybe I missed it?
- jfarlow 9y ago
  I understand your hypothesis that Cas9 only kills the cells that do not have the particular mutation and itself is a negative selection against the undesired genetic sequence. I do not believe the basic numbers and statistics of the rate of toxicity, size of genome, precision of mutation, etc. work out to permit that hypothesis, but it is a curious thought. As a hypothesis it is straightforward to test with a FACS-sorted population of cells expressing a fluorescent Cas9: does the fluorescent population die, or does the fluorescent population survive and contain the expected genetic mutation. That experiment has been done and is actually written up here as a protocol to more effectively using Cas9. The paper clearly refutes the above hypothesis and demonstrates that modifications by Cas9 are generally proportional to the amount of expressed Cas9 up to the point of toxicity [1]. In it they tag Cas9 with a fluorophore, express the construct in cells and sort for those cells that express Cas9 (separating them from cells that do not), and binning the cells based on the level of Cas9 expression. Given your hypothesis, those fluorescent cells should die and not have the desired mutations, while the non-fluorescent cells will be enriched for particular mutations not found in the fluorescent cells. Some toxicity is shown at very high levels of Cas9 expression, but the on-target edits occur in cells expressing Cas9, and those cells live for a number of passages. See Figure 3 in the paper below: > Cell population 1 displays GFP fluorescence intensities similar to those of nontransfected cells (autofluorescence level), whereas populations 2–6 display specific GFP levels ranging from low to high (varying ~500-fold). (e) 20,000 cells were isolated from each of populations 1–6 in (d) and cultured for 6 d, after which IDAA was used to determine indels at the KRAS locus in an aliquot of the cells. Note that editing levels increase with increasing GFP fluorescence level at the time of FACS and that almost complete editing was obtained at submaximal fluorescence levels... [T]he total number of live cells in each population analyzed in (e) was determined by trypan blue exclusion and expressed as % of population 1. [1] https://www.nature.com/articles/nprot.2016.165 https://www.nature.com/articles/nprot.2016.165 Given the above result, a significant amount of research is being done right now to preserve the useful properties of Cas9 - being able to home in on a particular genetic sequence - while engineering the protein to minimize and even prevent those deleterious cuts do DNA. And that research is finding steady success.
  - nonbel 9y ago

nonbel 9y ago

>"Wild-type Cas9 cuts genomic DNA, and when it does, the strand can go flying - with repair a possibility rather than an inevitability." "Possibility" is too strong a term. Afaict, the best you can say about repair is that it is a "less than common occurrence," and even that may be too much. >"Recent papers from the Liu lab at Harvard [1] (among many others) show how you can edit DNA using a modified Cas9 that has been modified to not have that second cutting function, while retaining the ability to bind to a specific DNA sequence." Sorry but I could not find any comment/data about toxicity in this paper. This is basic info everyone wants to know: What percent of cells exposed to this stuff die? Maybe I missed it?

jfarlow 9y ago
I understand your hypothesis that Cas9 only kills the cells that do not have the particular mutation and itself is a negative selection against the undesired genetic sequence. I do not believe the basic numbers and statistics of the rate of toxicity, size of genome, precision of mutation, etc. work out to permit that hypothesis, but it is a curious thought. As a hypothesis it is straightforward to test with a FACS-sorted population of cells expressing a fluorescent Cas9: does the fluorescent population die, or does the fluorescent population survive and contain the expected genetic mutation. That experiment has been done and is actually written up here as a protocol to more effectively using Cas9. The paper clearly refutes the above hypothesis and demonstrates that modifications by Cas9 are generally proportional to the amount of expressed Cas9 up to the point of toxicity [1]. In it they tag Cas9 with a fluorophore, express the construct in cells and sort for those cells that express Cas9 (separating them from cells that do not), and binning the cells based on the level of Cas9 expression. Given your hypothesis, those fluorescent cells should die and not have the desired mutations, while the non-fluorescent cells will be enriched for particular mutations not found in the fluorescent cells. Some toxicity is shown at very high levels of Cas9 expression, but the on-target edits occur in cells expressing Cas9, and those cells live for a number of passages. See Figure 3 in the paper below: > Cell population 1 displays GFP fluorescence intensities similar to those of nontransfected cells (autofluorescence level), whereas populations 2–6 display specific GFP levels ranging from low to high (varying ~500-fold). (e) 20,000 cells were isolated from each of populations 1–6 in (d) and cultured for 6 d, after which IDAA was used to determine indels at the KRAS locus in an aliquot of the cells. Note that editing levels increase with increasing GFP fluorescence level at the time of FACS and that almost complete editing was obtained at submaximal fluorescence levels... [T]he total number of live cells in each population analyzed in (e) was determined by trypan blue exclusion and expressed as % of population 1. [1] https://www.nature.com/articles/nprot.2016.165 https://www.nature.com/articles/nprot.2016.165 Given the above result, a significant amount of research is being done right now to preserve the useful properties of Cas9 - being able to home in on a particular genetic sequence - while engineering the protein to minimize and even prevent those deleterious cuts do DNA. And that research is finding steady success.
- nonbel 9y ago