I wanted to knock out MSH2 in mice cell line CH12F3 and after gRNA construction, transfection and single cell sorting, I got single colonies. I used two guide RNAs to give a deletion but when I checked the single colonies through PCR there were no deletion. Then rather proceeding with T7 assay, I grew the cells and tested for protein expression by WB. Also I analyzed the mRNA expression by RT PCR. Both were negative. Now I need to sequence to see the indels. Is it necessary to clone them into a vector or can I sequence my PCR products?
Hi Lakshani,
Assuming you are going for Sanger Sequencing for the whole population (not single clones) , it is still possible to directly sequence the PCR products and get an idea of indel percentage using softwares like TIDE. If you want a clean trace for you sequencing, then you would have to clone your PCR products into a vector and sequence at least 10 bacterial colonies to get an idea of indel percentage.
However, if you have already screened around 10-20 single colonies, and did not find any change in protein/mRNA expression in any of them, I would recommend checking if everything else is in working order, i.e. Cas9 expression, gRNA efficiency, transfection efficiency. If your gRNA efficiency is good, you should be able to detect decrease in protein expression even at the whole population level. If not, then it may not be worth going for clonal isolation because you will have to screen many many colonies before you find one that has a complete KO. If the system is not very established in your lab, it might be better to check for gRNA efficiency at the whole population level first, and then go for single clone isolation.
Hi Ayon,
Right now I have single clones with no protein expression and mRNA expression.
Hi Lakshani,
Sorry I misunderstood your question. In that case, yes it is possible to directly sequence your PCR products or cloning them into a vector. Cloning into a vector and sequencing individual colonies will be much cleaner of course. If you do direct sequencing, assuming there are two copies of MSH2 gene in the cell line, you will probably not get a clean trace in your Sanger chromatogram. You may try TIDE to decode the chromatogram, I personally found it very useful.
Hi Lakshani,
Abm has a good and informative page on CRISPR screening. You may refer to section 4.4 for screening indels:
https://www.abmgood.com/marketing/knowledge_base/CRISPR_Cas9_Screening_Validation.php
Section 4.4.2 has a list of programs that can help you with sanger sequencing directly from PCR products.
Hope this helps!
Hi
In direct sequencing of PCR product, sometimes the primers used for PCR may not work for sequencing reaction. Cloning the PCR product in a TA vector can provide an advantage of using universal primers (companies have all type of universal primers and their Tm standardized) for sequencing. However, if your primers are working in sequencing reaction, then go ahead with good quality PCR product.
If there will be few bp indels in the targeted region, there are chances of not getting fragment shift in even 3% agarose gel. In my experiment, i did not get any shift in PCR product, however, using the same PCR products, i found cleaved DNA fragment in 1.5% agarose gel after T7EI assay and that too in the region encompassed by sgRNA1 and sgRNA2. T7EI reaction gives a first estimate of the probable site of indels in the targeted sequence, which you can further match with the pattern of indels in sequencing result.
Thanks
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