Cells edited using CRISPR–Cas9 injected into a person for the first time • Hacker News Board

Re: Cells edited using CRISPR–Cas9 injected into a person for the first time

#51

Post by nonbel » Wed, Nov 16, 2016, 9:07 PM UTC

ihodes wrote: post #50

nonbel wrote: post #42

Earlier quoted context omitted.

>"CRISPR is used to mess with a gene, not kill a population of cells." Look it up, in every paper where they report on that aspect, the vast majority of the cells die. Also, they detect small numbers of mutants in the control (non-"modified") cells.

I haven't read a ton of CRISPR/CAS papers, but I have read the seminal ones, and didn't see that. Mutations in unmodified cells are completely expected, of course, but not mutations in line with where CRISPR is being targeted—I'd hardly believe that to be the case. It looks like the cell perforation (getting CRISPR/CAS into the cells) can certainly affect cell viability, but those numbers have been steadily, rapidly…

Looking at that paper, the 80% viability doesn't refer to after CRISPR/Cas9 had time to do the selection, it refers to passage through their chip and puromycin selection (which just indicates the plasmid got in the cell):

"Plasmids encoding Cas9 and sgRNA targeting phosphatase and tensin homolog (Pten) (fig. S6A) were delivered into MCF7 cells, followed by culture for 48 hours and puromycin selection. More than 80% of the cells survived the selection process, indicating the high delivery efficiency of our method.

[...]

Similar to Pten knockout, more than 80% of 53BP1 knockout cells survived the selection process."

You have to look at figure 5B, where the caption reads:

"Cells (5 × 10^4) from (A) were seeded in 60-mm dishes in complete medium and cultured for 7 days. Cells were trypsinized and collected for cell count in a Countess II FL Automated Cell Counter (Life Technologies) daily for 7 days."

So they started with 5 x 10^4 cells but if you look at the first day after being in culture, it is much less than that. I can't tell how low from the chart which shows it as basically indistinguishable from zero. They do not seem to report those values anywhere.

Also, you can look at figure S5 and see that they saw GFP get knocked out in >2% of control cells (Cas9 only). They don't do a similar quantification for the Pten, but if they had, I am pretty sure some low percentage of cells would be shown as already knocked out for Pten.

Re: Cells edited using CRISPR–Cas9 injected into a person for the first time

#52

Post by tropo » Wed, Nov 16, 2016, 9:12 PM UTC

dekhn wrote: post #48

tropo wrote: post #18

Earlier quoted context omitted.

Project idea: The death cap mushroom is really tasty according to people who are now dead. Disable the poison.

you would do that chemically, after collecting the mushroom/while cooking it, because that's easy.

not for the death cap

The poison is a bicyclic polypeptide that shuts down ribosomes in your liver. Destroying or removing the poison is as difficult as destroying or removing all the protein. (like dealing with mad cow prions) It's just not going to happen unless you like your mushrooms charred pure black all the way through. Well, that or dusty white ash.

Just half a mushroom will kill an adult human. This isn't something to take chances with.

Re: Cells edited using CRISPR–Cas9 injected into a person for the first time

#53

Post by nonbel » Thu, Nov 17, 2016, 1:02 AM UTC

nonbel wrote: post #51

ihodes wrote: post #50

Earlier quoted context omitted.

I haven't read a ton of CRISPR/CAS papers, but I have read the seminal ones, and didn't see that. Mutations in unmodified cells are completely expected, of course, but not mutations in line with where CRISPR is being targeted—I'd hardly believe that to be the case. It looks like the cell perforation (getting CRISPR/CAS into the cells) can certainly affect cell viability, but those numbers have been steadily, rapidly…

Looking at that paper, the 80% viability doesn't refer to after CRISPR/Cas9 had time to do the selection, it refers to passage through their chip and puromycin selection (which just indicates the plasmid got in the cell): "Plasmids encoding Cas9 and sgRNA targeting phosphatase and tensin homolog (Pten) (fig. S6A) were delivered into MCF7 cells, followed by culture for 48 hours and puromycin selection. More than 80% o…

>"knocked out in >2% of control cells"

That should be 1%.

Re: Cells edited using CRISPR–Cas9 injected into a person for the first time

#54

Post by dharma1 » Thu, Nov 17, 2016, 10:23 AM UTC

Ralfp wrote: post #39

dharma1 wrote: post #38

I have a feeling this won't stop at medical use cases

Most of technology is neither good or evil, and has usages in both civilian and military space. That is how the things are with technological progress. Machine learning may be used to find corelation between gene mutations and cancer, but it may also be used to put missile into unsuspecting enemy. Roman road system allowed for flow of goods and people within empire on scale never before seen in acient world, but also…

That's true. But I'm not talking just about military use.

I'm talking about germline gene editing, beyond gene therapy.

It will have consequences that span multiple generations, and are self-replicating. It will completely change the fabric of society. We are going to be changing what we are as life forms, without really understanding life in the first place.

http://www.nature.com/news/don-t-edit-the-human-germ-line-1....

Re: Cells edited using CRISPR–Cas9 injected into a person for the first time

#55

Post by dekhn » Thu, Nov 17, 2016, 2:29 PM UTC

tropo wrote: post #52

dekhn wrote: post #48

Earlier quoted context omitted.

you would do that chemically, after collecting the mushroom/while cooking it, because that's easy.

not for the death cap The poison is a bicyclic polypeptide that shuts down ribosomes in your liver. Destroying or removing the poison is as difficult as destroying or removing all the protein. (like dealing with mad cow prions) It's just not going to happen unless you like your mushrooms charred pure black all the way through. Well, that or dusty white ash. Just half a mushroom will kill an adult human. This isn't so…

Right, my protocol would have been: fully homogenize the mushroom, treat it with an antibody known to disable all the toxins, then cook and eat. I wasn't really proposing anybody does this.