Homology Arms for Knock-In via HDR

[Laura]

Hello, 

I am designing an experiment to knock-in a gene at a specific location in the mouse genome. 

The goal is to use HDR with dual nickases to create a specific cut. We will be using a plasmid vector to supply the gene of interest to the cell. The gene of interest is a little longer than 500bp, so to my understanding, it is necessary to have homology arms that are greater than 800 (~1000)bp long. 

Should the homology arms be encoded within the plasmid vector? Should they start aligning exactly at the location of the cut? 

Additionally, for primer design: should they be complementary to the gene of interest or the same bases as the homology arms are?

Thank you! 

[Julia Joung]

Hi Laura,

In my experience, homology arms that are ~850bp work best. The homology arms should be encoded within the plasmid vector and start aligning exactly at the location that you would like to insert the gene of interest, i.e. your plasmid vector should look like the insertion you would like. Of course, this should be as close as possible to your sgRNA cut site. The sgRNAs should be complementary to the genomic sequence at which you are trying to insert your gene of interest. This can be either the sense or the antisense strand, and both have worked for me.

Best,

Julia

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