Hi everybody,
I'm trying to perform CRISPR-Cas9 editing on an iPSC line in order to correct a single nucleotide mutation. I'm using ribonucleoprotein complexes (RNPs) to the delivery of the Cas9 along with an ssDNA oligonucleotide repair template to achieve the homology directed repair.
I have already repeated the experiment several times and I have only obtained heterozygous clones. I don't know why I can't get an homozygous clone. I have sequenced the iPSC line and I have not found any SNP in the NGG nor in the sgRNA.
I'm stuck at this point because I don't know how to solve this problem.
Can anybody please give me some advice?
Thank you so much!
It is possible that you're targeting an essential gene. Lists of commonly essential genes can be found at the Depmap site.
Check also if you are targeting a gene that is expressed monoallelically..you might be preferentially targeting the active allele..
Kristian Bruun Laursen Andrea Cerase Thanks for your answers! I'm targeting PYGM (the muscle isoform of glycogen phosphorilase), as far as I know this is non an essential gene and it's expression is biallelic, so this might not be the problem.
Try using a combination of two-three similar guides to increase your chances then. I guess a double hit happens less frequent than a single hit . The other way is to add a selection casette and selection, but that's not always possible.
PS to be really sure of biallelic expression, of your gene, you either need to use nascent FISH, or a technology that relies on SNPs/Polymorphisms (if you have any), otherwise you will never be sure, regardless what is reported in the literature. Most genes get switched on in bursts of transciption followed by a transcriptional off state, and not always both alleles are expreesed at the same time..
A way around the problem could be to use the hets (with one repaired allele). Targeting specifically the undesirable allele should facilite biallelic corrections (even without exogenous provided template).
Andrea Cerase You are right, i'm assuming that it's expression is biallelic because of the literature and furthermore, in my lab, we have already edited another mutation of this gene in homozygous (so I expect that this wouldn't be a problem). I'm considering using another sgRNA in the next experiment to see if it increase the chances.
@Kristian Bruun Thanks, that was one of the approachs that I was thinking about.
I read the recently published paper following the attached file. They established a synergistic gene-editing protocol that generated precise genetic modifications in human iPS cells. It may be useful for your work.
I understand how difficult it is to obtain homozygous colonies. I recently attempted to delete the 15Kbp gene and ended up with several heterozygous colonies for nearly two years of multiple tries. Then I planned and re-targeted heterozygous colonies by changing the drug selection cassette on donor arms. After nearly two years of trying, I finally had success with only one colony for homozygous. I hope to re-targeting on heterozygous colonies help. Thank you
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