Problem with knock-in experiment (px458, ssODN)

I am a newbie in Crispr and now I have a problems with knock-in experiment.
Currently I am trying to get knock-in cells with one missense mutation.
For my first initial experiment I've designed ssDNA oligo as donor template with homology arms (left: 42bp; right: 38bp; total lenght of template: 100bp). I added two silent mutations (one mutation in PAM and second mutation that form new restriction site). I ve desined guide sequence by using "crispr.mit.edu" tool and cloned into the pSpCas9(BB)-2A-GFP (pX458) vector. Then I performed transfection of px458-Cas9 (2ug) and ssODN into HeLa cells by using Effectene reagent. I used ssODN in two different conc.: 300ng and 3000ng.
I tried this experiment twice and result was unsuccesful. I got only KO cells with mutations near PAM. Also I tried to use plasmid (pcDNA3.1 with conc. 0.5ug and 2.5ug) as donor template. Result was same.
Could you please tell me where I made a mistake.


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Green Mummyover 3 years ago

Based on the information you gave to us, you are probably not doing anything wrong. The fact is the efficiency of HDR is low. I would suggest you to first verified the presence of genome edit in the pool of transfected cells by T7 assay. Later, if you inserted an endonuclease site in your ssODN, you can verify HDR by a simple RFLP assay, and then start to isolate clones.

Furthermore, you can increase ssODN length and follow other suggestions as described by Paquet et al 2016, Nature (https://www.nature.com/articles/nature17664). Or, https://blog.addgene.org/optimizing-donor-dna-for-enhanced-crispr-genome-editing

Good luck and please let us know what gave you better results.

Khaki Yetiover 3 years ago

did you try to add phosphorothioate internucleotide linkages in the first 2 and last 2 nucleotides of your ssODN repair?

It has been a game-changer for me in the past.

Olive Ghostover 3 years ago

Our lab was having similar problems getting HDR in HL-60 cells. We found that treating for 24h with 25ng/mL nocodazole massively improved our HDR efficiency. We recently published a paper on our work:

https://www.ncbi.nlm.nih.gov/labs/pubmed/30670178-establishment-of-a-f508-cf-promyelocytic-cell-line-for-cystic-fibrosis-research-and-drug-screening

I hope this suggestion helps!

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