I would suggest using two different guides flanking the 1kb you intend to take out. Then you'd only need to design the donor to have 800bp arms homologous to the regions flanking the 1kb excised sequence.

In C. elegans, 800bp homology was  enough to insert GFP, which is around 1kb (Kim et al Genetics 2014).

Some important points to keep in mind when designing your experiment is the organism you're working with and whether or not there's an efficient way to screen for positives (antibiotic resistance or GFP for example). If you're working with an organism with a short life cycle, you could also break up the process: First confirm that the unwanted sequence has been removed, then proceed with insertion. If you're working with an organism that gives plenty offspring and if you have a way to screen for insertion, then do it all at once and search for positives.

Thank you Ahmed,

I will be using two different guides to excise my strand and I gather the activity of one sgRNA does not impede the activity of the other, but I'm still unsure of the insert strand.

What would be the biggest region that I can insert? Would I be able to insert multiple genes in this region? And if I do, is there a scale of homology arm length to length of insert or is the general length 800bp per arm?

I will be doing this in cells and I'm designing this with a screen too, so it'll be single step.

Hi Martino,

Mega inserts occur in nature resulting in major chromosome rearrangements. The fact that you are using cells is an since you'd be able to screen for rare events. My impression is that the bottle neck here is your ability to make a construct with the large insert and transfect it in your cells. I don't think that the problem is the homologous recombination as much as it is the construct and delivery.

Your question about the scale of homology arm length in relation to insert is a good one, and what I have experienced discussing this with colleagues is that we assume that the larger the insert, the longer the homology arms. Usually, this leads to an exaggerated length that is then reduced. I would suggest that 1kb homology per arm is sufficient, even if your insert is 10kb. In that sense, why not start with a working assumption that the homology arms should be one tenth the length of the insert.

I hope this is helpful.

Ahmed

I have successfully deleted about 5kb and then inserted around 4 kb via HDR in erythroid cell lines. I used TALENs in this approach but that should not make a difference! My arms of homolgy were about 1kb to each side!

Good luck!