So, I'm about to start this new project and I'm wondering if there are new trending techniques to insert LoxP sites flanking an exon in the mouse genome. We use CrispR/Cas9 system in the lab, so my idea is generating a plasmid with two big homology regions at each side (around 1.5Kb), then the floxed sites and the exon. Then, injecting that along a guide that will cut somwhere in the middle of this region just to allow recombination.
What do you think? What are your recommendations? Is there a new way to insert LoxP sites? They will be about 2.7 Kb apart since the exon I'm trying to get rid of is quite big.
Also, what is your experience with the different LoxP sites? I've been looking for the sequence and found there are many, does the effectiveness of the recombination depend on the sequence of the LoxP site? Does it change depending on which Cre you are using? If so, what is the best LoxP site?
Thank you very much in advance for your time and patience. I'm super new to this area so any information will be of a lot of help.
Hi Daniela. The way you suggested for inserting the loxP sites using CRISPR and a donor is still the best way. 2.7 kb is not very big for these donors. alternatively, you could probably insert each one using paired nickases and dsDNA donors, although that sould be less efficient. GeneCopoeia has made many such donors and can help you with this type of project, including the heterologous loxP sites. To learn more, visit our CRISPR page: http://www.genecopoeia.com/product/crispr-cas9/
Hi Daniela.
Hope your project has already taken off, but if not, here are a few key points:
The main challenge with HDR using long repair templates is to distinguish correct from random integrations, and the larger your HRs are, the larger a region you would need to amplify using a pair of primers, which are specific for template and genomic region (outside the HRs). In orther words; templates with smaller HRs are less efficient, but easier to confirm. That said, a primer pair spanning the integration site would allow for easy detection of homozygous knockin. The problem is that you might not get any.
Also make sure that your CRISPR cannot target your repair template (e.g. by mutating PAM and/or targeting sequence).
See also this publication https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938658/
And feel free to contact me with questions.
Best, Kristian
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