I am using CRISPR Cas9 to introduce a point mutation in a gene to get patient-like cells. I am applying a plasmid with cas9 and my gRNA inserted. I used a ssODN as a template for introducing the desired mutation. After puromycin selection and limiting dilution to get single-cell clones and after almost two months when I get the sequencing results it seems that there are no indels etc, it seems that there is no double-stranded break in the desired site in the genome.
I am not aware of a faster method than what you are doing except doing some very hardcore biochemistry. Presumably you used a design tool to generate your gRNA. It's possible that your gene is being selected against and that your particular cell line cannot survive without this gene, so you are not able to isolate any KOs from it.
Connor Jankowski Actually I just want to introduce a point mutation in CEP290 gene in HEK293T cells. I am not doing any knockout. I have used two different gRNAs but nothing really happened as all of the clones I get after puromycin selection there is no indels or any other mutations.
Did you use the donor to introduce the altered the point mutation? so that's a Knock-in?
Azhar Zeb. I understand the hard waiting of two months just to know that your experiment has not worked. Generally, people expect the selected clones to have a deletion but to be more precise these are two events. Having a positive clone only means that the selected cells had taken up the construct and are more likely to express your guide RNA sequence towards the target sequence of the genome. also the expression of sgRNA sequence doesn't necessarily mean that the sequence will make a deletion. ( there is varying efficiency and accuracy of gRNA sequences towards the target sequence) and algorithms used by online tools we use to design the gRNAS are predictable scores .( because there are certain lot others factors we are yet to understand affecting the accuracy and target cleavage efficiency of gRNA)
so to put in a simple way and to answer your question we generally start by cloning 3-4 guide RNA sequences against a particular gene/target sequence(to increase the probability).
and to not wait for the sequencing results we isolate the DNA from transfected and selected cells after 48-72 hrs (we use puromycin). we go for the T7 endonuclease assay. T7 endonuclease assay will help you understand if your sgRNA sequence has been able to make the deletion and the efficiency of the deletion. (kindly go through the principle of the technique you will understand and if you need any assistance in terms of protocol you can reach me @ [email protected] I can help you with the optimised protocol)
after that, you can use the genotyping primers to PCR amplify the gDNA of target cells and further confirm by the sequencing.
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