Donor DNA design for 3x Myc insertion

I want to insert 3x Myc into the C terminal of my gene in A549 or/and SV589 cells. The tag will be 114 bases. I have several basic questions about donor DNA:

1. How long is enough for the homology arms on each side?

2. For that 114 bases insertion, what type of donor DNA is better, ssDNA, dsDNA or a plasmid?

3. What's the transfection amount of donor DNA?

Hope that your experienced CRISPR/Cas people can give me the answers.

I really appreciate your help.

Yaqin


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Gold Zombieover 4 years ago

For a sizable insertion, a couple hundred nt homology arm should work well; see e.g. Fig. 2 in this paper. I'd recommend using a long ssDNA donor. Linearized plasmid is undesirable because it will integrate into unintended locations in the genome of your cell line, and plasmid donors can have fairly low efficiency. For donor synthesis, you can use a commerical source (e.g. here) or in-house production (e.g. here). Concentration can be optimized depending on your cell line but generally higher is better if the cells can manage it. If the goal is to get a homozygous line, to maximize efficiency it's a good practice to select the guide so that the cleavage site of Cas9 is as close as possible to the insertion location (see e.g. here).

Peach Garudaover 4 years ago

You should be able to get away with 50bp of homology either side for an insertion of this size. Or, try 91bp + 36 bp to give asymmetric arms (our preferred protocol). This gives an oligo of under 250bp. IDT may synthesise this for you. If so, this would be much easier than lssDNA

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