Recently, I used CRISPR-Cas9 to knock out a gene. The cells were double selected by Blasticidin and puromycin. The clonal cells were detected by immunoblotting which the target protein was expressed much weaker compared to the parent cells, but it is still a tiny band. I did QPCR to further confirm the immunoblotting result. After calculation, the target gene in the clonal cells were only less than 10% of parent cells.
Does CRISPR-Cas9 knock out gene remain another strand of DNA which may still express at low level?
HI....
Recently, I used CRISPR-Cas9 to knock out a gene. The cells were double selected by Blasticidin and puromycin. The clonal cells were detected by immunoblotting which the target protein was expressed much weaker compared to the parent cells, but it is still a tiny band. I did QPCR to further confirm the immunoblotting result. After calculation, the target gene in the clonal cells were only less than 10% of parent cells.
Does CRISPR-Cas9 knock out gene remain another strand of DNA which may still express at low level?
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CRISPR-Cas9 is a powerful tool for genome editing that allows researchers to make precise changes to the DNA of living cells. When used to knock out a gene, CRISPR-Cas9 can be very effective at reducing or eliminating the expression of the targeted gene. However, in some cases, a small amount of the targeted gene may still be expressed even after CRISPR-Cas9 treatment.
There are several potential reasons for this:
CRISPR-Cas9 may not completely eliminate all copies of the targeted gene. If the gene has multiple copies in the genome, CRISPR-Cas9 may only knock out some of them, leaving others intact and functional.
CRISPR-Cas9 may cause off-target effects, resulting in unintended changes to the DNA that may affect the expression of other genes.
Some genes may have multiple promoters or transcriptional start sites, and CRISPR-Cas9 may not eliminate all of them, resulting in residual gene expression.
The knockout may not be complete, and the gene may still be expressed at low levels due to incomplete degradation of the mRNA or protein.
In conclusion, it is possible that CRISPR-Cas9 may not completely eliminate the expression of the targeted gene, resulting in a small amount of residual expression. To confirm the effectiveness of the knockout, it is important to use multiple methods, such as immunoblotting and qPCR, to carefully analyze the expression of the targeted gene in the treated cells.
I hope this information is helpful.
Hi Chunfa Huang
It seems you are targeting a protein coding gene, I could think of the followings
1- What's the design and strategy for targeting your gene of interest? (eg.. did you target specific coding domain, all isoforms, did you create a frameshift? what is the immunogen sequence of your antibodies?
2- You have to make sure your selected clone is homozygous (with genotyping PCR and sanger sequencing)
3- the antibodies might not be specific or they may be an alternate isoform ?
4- redesign a qPCR primers to the target exon and try to amplify it? it should be at least 10 cycles higher than WT for complete KO
5- your clone might be heterozygous?
6- I am happy to discuss more if needed
I hope this help
Amr
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