You could possibly get upto 300bp from what has been reported in the literature. Ideally you would have separate selections for each individual gRNA to increase your chance of finding that both cuts have been successfully induced.

I'd say 300bp is a very ambitious deletion, maybe consider use of a template to increase the efficiency and chance of actually inducing the desired deletion - if this is to be bi-allelic as well then the more prep work you do before actually CRISPR'ing the more you will increase your chances of success.

Also I'm really curious to hear other peoples experiences on this!

Good luck :)

https://www.nature.com/articles/s41586-018-0380-z

Cengiz Akbulut

Thank you so much for the information !

300 bp was much shorter than I have expected.

Actually, I'm planning to knock-in an inverse puromycin resistant gene which is flanked by 5' and 3' arms using a targeting vector.

In this situation, do you think the maximum size of the sequence you consider to delete would differ?

Interesting, so you're planning on using CRISPR to insert, or knock-in several kilo base-pairs to your cells. This is very do-able and reported by various ways, resources available on AddGene etc.

In my current project I am doing this with an expression vector, resistance genes etc. In total about 5000-6000 base pairs added to my cells. Nothing inventive, just applying existing techniques to my research question!

This is a bit different from a deletion where I thought you'd want to just remove a large chunk of genomic DNA from your target.

If you have a paper to recommend where they 'delete' more than 300bp I'd be super curious as to the method :)

Cengiz Akbulut

Thank you again for your answer.

I'm sorry to have confused you. I guess my explanation was not appropriate.

I'm trying to delete about 5 kb of DNA from my target and insert Puromycin resistance gene just for following seleciton.

I'll be done by co-transfection of 3 vectors, which are 2 different sgRNA–harboring pX330 vectors and one Puromycin resistance gene–harboring targeting vector.

The Puromycin resistance gene, which is about 1 kb, is flanked by 2 arms of DNA having the same sequence as both sides of the gene I intend to delete.

I just wondered that if it's a problem when the two parts of DNA are 5 kb away from each other after the cut by CRISPR/Cas9 system, and if I should reconsider about which part of DNA to delete.

I think this is a very primitive question.

Thank you very much for your time to answer.

The largest deletions I have personally made were about 10kb (in stem cells), but I've seen other reports that have done megabase locus replacements. With puromycin, it will be easier to do the selection, but you will have to screen to make sure that you have done a replacement, and not a simple insertion, and to also screen for random integrations (the size of your insert becomes an important factor when designing your screening approach). Also, from your scheme, the orientation of your puroymycin cassette is not that important, so a "homology-directed" scheme is not essential (maybe important for the previously mentioned point).

Without seeing the constructs and the targets, I can't give more advice (ie, will you be simultaneously cutting the plasmid, one or two cuts, orientation of the cut sites, single strand cuts versus double strand cuts, etc, etc)

All that being said, what you propose is certainly doable as long as you have the proper screening.

Nicolas Kuperwasser

Wow, I have missed this in the literature. When searching CRISPR deletion, couldn't find anything more.

Do you have this 10kb deletion in a publication? How did you do it? Did you 'just' cut at two ends of your target site and the 10kb 'excised' itself out?

Nicolas Kuperwasser

Thank you so much for the information :)

It seems not to be so hard to do what I'm planning. I'm pretty relieved.

Now I need to make sure that I can screen the right clone.

Thank you again.

Cengiz Akbulut Hi! I just have read your answer about inserting 5000-6000bp to the genome. I only have found papers where they describe knock-in of maximum 3.7 Kb. Can you please cite those papers where they describe larger amounts of dna inserted, please? It would be fantastic, thank you.

José Javier Marqueta-Graica

My mistake with this directly. Its not quite that you use CRISPR to insert fragment, they use zinc finger nucleases in this paper to insert something of this size.

Hope it helps!

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