Hello,
I want to target a specific gene sequence using CRISPR-Cas9 to induce the death of the cells carrying that gene. I think that there are three basic approaches: 1.) Induce a frameshift or irreparable chromosomal abnormality 2.) Confer expression of a pro-cell death gene 3.) Confer transcription of an siRNA against a pro-survival gene. Has this ever been done before? What are some basic technical considerations for using CRISPR-Cas9 to selectively kill?
I think that the most simple way to do this would be to induce a frameshift mutation. However, will mammalian cell lines in culture which are transformed or from cancer even be susceptible to killing by a frameshift? How does one insure that frameshift will or will not occur?
In my experiment, another cell line without the gene for targeted killing will be the control. That cell line should not die when Transduced with the CRISP-Cas9 virus.
Thank you!
Well, you can target the BIRC5 Gene also as they did in the article below.
Article Knockout Of BIRC5 Gene By CRISPR/Cas9 Induces Apoptosis And ...
However, there are other induced cell death strategies by using CRISPR/CAS9.
Chapter Using CRISPR/Cas9 technology for manipulating cell death regulators
It's a little hard to visualize your question. If you want to detect whether a frameshift has occurred, you can sequence the region targeted by your sgRNA(s). There are online tools like Synthego's ICE analysis that let you compare a parental sequence and edited cells to evaluate indels. However; if you want to ensure that a frameshift has occurred, you should perform single-cell dilution so that you have clonal cell growth before sequencing. If you don't, you will always have a mixture of cells with different kinds of edits and no edit at all. In this case, it won't really be possible to determine specifically whether a frameshift occurred, although you could evaluate the overall impact of your gene editing by performing immunoblot on your edited cell mixture and the unedited (or control) parental cells to see the degree to which the edited cells have lost expression of your target.
As far as lethal genes to delete, there are many; but if you target a gene that causes lethality when lost, the edited cells will quickly die and be hard to recover for analysis to really confirm that they are dying from the frameshift.
Last thought - always good to identify a target gene that has been published in your cells so it is known to be appropriate for your context. Genes often have different functions and dependencies depending on the type of cells in which they are present!
-all of this is assuming you are doing CRISPR-Cas9 + sgRNA editing based on NHEJ (random). If you are wanting to use HR repair, it is a separate set of difficulties
In this case, if you target a gene with main role in apoptosis induction, it will be difficult to culture and proliferate cells as they are innately apoptotic.You also have to knock out both alleles as homozygous. However, this paper "10.2147/BLCTT.S230383" reported that survivin knockout results in apoptosis.
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