3 ms·

I am wondering how they know these mutations were not present to begin with. Also, how many cells were there before the CRISPR treatment vs after? What was the

by nonbel 11y ago

I am wondering how they know these mutations were not present to begin with. Also, how many cells were there before the CRISPR treatment vs after? What was the rate at which they divide under these conditions? Perhaps they just killed off enough of the cells with the CRISPR/cas-9 treatment and it took a few days for them to recover to the point of producing detectable CA-p24 (an indicator of HIV) levels.

adrusi 11y ago
It doesn't matter if the mutations were there to begin with. Even when the mutation occurs after a change in the environment, it's not the change in the environment that caused the mutation, mutations are random. What matters is if the treatment made the mutations viable.
- nonbel 11y ago
  Sure it matters. They say that "all mutations cluster around the Cas9 cleavage site at position −3, suggesting that the escape mutations were generated in the process of HIV-1 inhibition." http://www.ncbi.nlm.nih.gov/pubmed/26796669 http://www.ncbi.nlm.nih.gov/pubmed/26796669 Perhaps instead the virus that infected some cells that was already mutated, so it was resistant to the cleavage (due to lacking the recognition site). These are totally different mechanisms.
tosseraccount 11y ago
Original paper is here : http://www.cell.com/cell-reports/fulltext/S2211-1247%2816%2930298-4 http://www.cell.com/cell-reports/fulltext/S2211-1247%2816%29... CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape Supplement here : http://www.cell.com/cms/attachment/2052606220/2059839343/mmc1.pdf http://www.cell.com/cms/attachment/2052606220/2059839343/mmc... Known strain to start with. They claim "Both viral targets are very conserved in HIV-1 sequences that are registered in the HIV database (Figure S1B)." See explanation of figure S1 in supplement for more info.
- nonbel 11y ago
  Ah, I was looking at the other paper so haven't inspected the one you linked to. The two papers used the same method though: "The HIV-1 LAI stock was produced by transfection of 293T cells with the pLAI molecular clone." "HIV-1 was first produced by transfecting HEK293T cells with HIV-1 DNA" Sounds like they produced a bunch of virus in 293T cells, during which time it could mutate.

by nonbel 11y ago

adrusi 11y ago

It doesn't matter if the mutations were there to begin with. Even when the mutation occurs after a change in the environment, it's not the change in the environment that caused the mutation, mutations are random. What matters is if the treatment made the mutations viable.

nonbel 11y ago
Sure it matters. They say that "all mutations cluster around the Cas9 cleavage site at position −3, suggesting that the escape mutations were generated in the process of HIV-1 inhibition." http://www.ncbi.nlm.nih.gov/pubmed/26796669 http://www.ncbi.nlm.nih.gov/pubmed/26796669 Perhaps instead the virus that infected some cells that was already mutated, so it was resistant to the cleavage (due to lacking the recognition site). These are totally different mechanisms.

tosseraccount 11y ago

Original paper is here : http://www.cell.com/cell-reports/fulltext/S2211-1247%2816%2930298-4 http://www.cell.com/cell-reports/fulltext/S2211-1247%2816%29... CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape Supplement here : http://www.cell.com/cms/attachment/2052606220/2059839343/mmc1.pdf http://www.cell.com/cms/attachment/2052606220/2059839343/mmc... Known strain to start with. They claim "Both viral targets are very conserved in HIV-1 sequences that are registered in the HIV database (Figure S1B)." See explanation of figure S1 in supplement for more info.

nonbel 11y ago
Ah, I was looking at the other paper so haven't inspected the one you linked to. The two papers used the same method though: "The HIV-1 LAI stock was produced by transfection of 293T cells with the pLAI molecular clone." "HIV-1 was first produced by transfecting HEK293T cells with HIV-1 DNA" Sounds like they produced a bunch of virus in 293T cells, during which time it could mutate.