I'm currently planning an experiment for knocking out PARP1 in a human cell line with either the Nuclease or Double Nickase system. I'm not sure which region of the gene would be the best to target at. I think it would be the most efficient to choose an exon but the question is which one.
1) Has anyone tips for a specific domain that could be a good target?
2) Has anyone already experience with knocking out PARP1 in a human cell line and could get me some tips which system he/her was using and which sequence was effective to reach a succesful knockout?
Thank you for your answers.
Hello Sabine,
for a "simple" knock out of any gene you want to choose an early exon (definitely not an intron!) (preferably your first translated exon) and try 3 - 5 different guides for the knockout. You want to use an early exon, as you'd like the DSB to be repaired by NHEJ, to cause a del/indel resulting in a frame shift, which will ultimately lead to a premature stop codon. This will most likely lead to no protein product being translated at all. Should you still get a truncated protein, it will be non-functional as you altered the sequence very early.
There are several online tools for selecting "good" guides (i.e. high target efficiency and low number of off targets). I currently use CRISPOR http://crispor.tefor.net/
If you get good guides in your target sequence I would definitely try the WT Cas9 first, and not the nickase. WT is much easier and more efficient in our hands.
Depending on your system you can do puro selection, and do western blot on the bulk cells to check for knock out, maybe + some NGS method to verify the induced mutations. Or you do single cell clones with western blot and sanger sequencing.
Hope that helps.
Hello Sabine,
for a "simple" knock out of any gene you want to choose an early exon (definitely not an intron!) (preferably your first translated exon) and try 3 - 5 different guides for the knockout. You want to use an early exon, as you'd like the DSB to be repaired by NHEJ, to cause a del/indel resulting in a frame shift, which will ultimately lead to a premature stop codon. This will most likely lead to no protein product being translated at all. Should you still get a truncated protein, it will be non-functional as you altered the sequence very early.
There are several online tools for selecting "good" guides (i.e. high target efficiency and low number of off targets). I currently use CRISPOR http://crispor.tefor.net/
If you get good guides in your target sequence I would definitely try the WT Cas9 first, and not the nickase. WT is much easier and more efficient in our hands.
Depending on your system you can do puro selection, and do western blot on the bulk cells to check for knock out, maybe + some NGS method to verify the induced mutations. Or you do single cell clones with western blot and sanger sequencing.
Hope that helps.
Thank your very much for your answer Thomas, that was really helpful.
Hi Sabine, in my experience it depends. So you can put your gene of interest to design gRNAs, the website will rank the gRNA sequences with scores, you can try from the highest scored sequences. Usually targeting the exons close to the promoter gets high scores.
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