The CRISPR nuclease vector contain an OFP reporter but is not encoded with antibiotic resistance gene.
24 hours following lipofection, there was no significant orange fluorescence under the fluorescent microscope (OFP has a peak excitation of 548nm, and emission of 560nm). This might be due to loss of fluorescence, instead of low transfection efficiency.
FACS enrichment was not feasible. Due to the lack of (if any) orange fluorescence, only few cells were positively sorted.
Western blotting demonstrated that there were only
Hi Melissa,
CRISPR/Cas9 knock-out is based on creating double-stranded breaks in the genome, followed by potential mistakes (deletion or insertion) of nucleotides due to mistakes made during repair processes, resulting in a frameshift. Since every amino acid is encoded by 3 nucleotides, removing 1 or 2 nucleotides would result in all the downstream amino acids changing, often resulting in the termination due to early stop codons.
Unfortunately, in many cases you can still get expression of your protein if you remove exactly 3 (or a multitude of 3) nucleotides because you are not causing a frameshift. Mind you, functionality, stability or trafficking could of course all be affected by your mutation - but it could also still be functional. Either way, you'll still pick it up on Western Blot and qPCR. And after this frameshift, no new recombination will work because your sgRNA no longer binds (part of the target sequence is now missing).
Basically, what this means is that only 2 out of 3 mutations cause a frameshift that will guarantee the knockout of your protein. That's 67%. Additionally, you often have 2 copies of one gene (maternal + paternal) where the same odds apply. 2/3 * 2/3 = 4/9 approximately 45%. So if a 3 / 6 / 9 etc. nt frameshift does not really affect your protein, on average around 2/9 of your cells will express half of the gene, and 4/9 will express none of it. So total expression levels would be (3/9 * 1) + (2 / 9 * 0.5) = 4/9 = approx 45%.
Altogether, it sounds like you're getting a pretty reasonable knockout. This is the reason why people:
A) Sort cells or select monoclonal cultures after CRISPR/Cas9 transfection to select for cells with complete KO. You are now sorting for cells containing your construct. Can you use antibodies to sort for cells negative for the protein you are knocking out? If not, you might have to expand monoclonal cultures and check their KO separately.
B) Use more than one sgRNA. If you choose an additional target sequence further downstream, statiscally your are improving your odds of creating a KO.
Your knockdown efficiency is really good. As Olivier explained, it this means that many 100% knockout cells are probably in there. You just have to isolate them.
Plate the cells out at extreme dilution, like 1:4000 for a confluent culture; Each individual cell gives rise to a colony of offspring around it that inherit its particular mutation. (this may take some weeks of culturing). Then, pick the colonies out out and grow them separately. Test the clones by PCR or western to find one that has lost expression altogether.
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