if so, which length approximately should each homology arm be?
Thanks in advance!
7.5 kb insert Is pretty large. I am not sure whether the length of the homology arm is to do with the length of the insert (ex. longer insert needs longer homology arms??). Here is the website provides some pointers. Please see (3) and (4) of the Homologous Recombination (HR) section, where Dr. Feng Zhang's Lab suggests using 0.8 kb as the homology arms.
https://www.addgene.org/crispr/zhang/faq/
At least in my experience in D. melanogaster, the standard for homology arms is 1kb on either side of your insert. However, everything I've read and everyone I've talked to about HDR insertions comes to the same conclusion: HDR rates are lower with bigger inserts. I've heard of 5kb knock-ins in mice, but I personally haven't heard of anything bigger than that so far. But I could just be behind the times.
I suggest designing your experiment with an easy, high-throughput screening method in anticipation of low efficiency. I'd also recommend doing some research of conversion tract lengths for plants to get an idea of how much homology you will need.
Best of luck!
@ Erwin,
Except homology-dependent knock-in, homology-independent knock-in is another choose. See this attached paper (2016), a near 5-kb fragment was efficiently knocked in through NHEJ repair pathway.
From the paper: "With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ireseGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells."
By the way, this group (2016) knocked in a '10-kb' fragment (containing metabolic pathways) into E. Coli with CRISPR (see attachment). You can check out and take a look.
I couldn't obtain the full paper. If any one of you can obtain one, please let me know. I would like to read it too.
Thanks,
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