Hi.
I want to knock-in 3xFlag-tag at the 3'end of my target gene, upstream of the stop codon. Hence, I will add the STOP codon after the 3xFlag-tag. Besides, I want to introduce LoxP-Sv40pro-Neo-LoxP for selection after transfection into mouse embryonic stem cells.
So the transgene cassete will be 3xFlag-(TGA)-LoxP-SV40pro-Neo-LoxP.
My problem is I might have poor sgRNAs design that can cut near the STOP codon. I used the crispr.mit.edu to find sgRNAs designs and pick 7 decent designs with highest score. I cloned them in pX330-U6-Chimeric_BB-CBh-hSpCas9(HF) respectively.
I did the EGFP disruption assay and 3 designs showed good fluorescent signal. I further check the sgRNAs using T7E1 assay by transfecting the sgRNAs into the mouse ES cells. But I could not get any cleavage while the positive control for T7E1 was working.
Currently, I'm trying to repeat the T7E1 assay which I suspected that my transfection efficiency could be affecting the result.
I also tried using CHOPCHOP sgRNA tool chopchop.cbu.uib.no/index.php which I think it provides better sgRNA analysis than the crispr.mit.edu. However, the best sgRNA from the tool is located downstream, around ~280 bp from the STOP codon.
As an alternative approach, i'm planning to design donor plasmid, which the design is illustrated in the figure attached.
I need to place the 3xFlag upstream the STOP codon, so is it possible to place the tag within the left homology arm itself?
Can I have successful knock in from this approach?
Meanwhile the Flox-SV40-Neo cassette will be added just downstream the DSB, followed by the right homology arm, which will be at the 3' UTR.
Hi.
I think that your design is possible in theory, but in practice, you might have to screen a large amount of clones before you finally find the right one.
The distance between the integration site and DSB site should be as short as possible. It is reported that successful point mutations were achieved ~100bp away from the cut site by ZFN, and the successful possibility of KI decreases when this distance increases.
Dr. Feng Zhang also recommends to design the KI site ideally 10bp from DSB site in his F&Q about using CRISPR-Cas9 mediated KI at Addgene.
About 1year ago, I tried to use both TALEN and CRISPR to knock a 3xFlag tag into my gene of interest, quite similar to your design, only differernt in the distance between the integration site and the cut site (~30bp in my design). Unfortunately after several rounds of experiment, I got no positive KI signal from the cell pools after antibiotic selection using gemonic PCR.
Until recently I used a non-HR mediated KI stratergy via CRISPR to knock 3xFlag and EGFP into my gene of interest. The KI site was designed to be just the cut site. Genomic PCR of the cell pools showed much higher KI effeciency and several positive KI cell clones were obtained for further sequencing analysis.
Hope my experience could help.
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