So we made a CRISPR mouse and got both loxP sites on same allele (no small feat). The 5' loxP site is perfect, the 3' loxP site, located 1.5 kb downstream, as lost an "A" nucleotide at the 5' end of the 34 bp loxP sequence.
Will Cre still excise or must I start over again or, wait a tick, maybe just genome edit in that missing "A"
SOmething for all doing CRISPR-Cas9 with loxP sites to ponder over.
Hopefully someone out there has tested such a mutant loxP site for functionality.
Thanks!
Joe
People have created some mutant loxP sites by replacing some of the 34-bp bases. These particular mutant loxP sites can recombine with a wild loxP site, and reduce the chance of 'reverse' reaction. If the 'A' locates at the middle of the 34-bp fragment, it might abolish its recognition. But, your 'A' is at the 5' end of the 34 bp loxP sequence. You might still get a chance to see it works.
As a follow up, we indeed found that our 1 bp deleted loxP still works in a cross with CMV-Cre. Thus, this 5' deleted base, located outside the Cre binding region, still retains recognition and Cre-mediated excision. There should be a resource for defining tolerable versus intolerable bp changes in loxP and other sites (e.g., Frt).
@Joseph,
Thanks for the update.
I think that people have done some experiments of that (tolerable versus intolerable bp changes) using replaced bases in the loxP sites. You can find some of them such as lox66, lox72,........etc online. They use these mutated sites for Cre-mediated 'integration' to trap the transgene in the integration site. But I did not find a database about it.
What is the efficiency of your 5' 1-bp deleted loxP for deletion?
By the way, did this 1-bp deletion naturally occur or man-made error? People have found att site (phiC31-att system) with nucleotide deletion in the transgenic lines.
Hi Yuan
This error occurred via NHEJ during CRISPR-Cas9 mediated genome editing. Efficiency is a hard one to answer since wtCre is not 100% anyway (at least in the mouse with a cell-restricted promoter). A review of the literature I did many years ago reveals ~80% of cells with excision. It remains to be seen what this loxP site will show but the fact that it excised at least tells us we will get some excision and not have to go back and make a new mouse with the corrected site.
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