Designing a 8kb replacement using HDR
I am new to CRISPR and have been doing deletions so far using NHEJ. We have a project that needs 5 SNPs in a span of 8 kb to be altered. So we are considering if we can replace the 8kb fragment using a donor DNA in a cell line. Has anyone done something like? Any suggestions are welcome.
My two cents:
1. To improve efficiency, I would include a selection marker, flanked by loxp/frt sites (put it somewhere in the intron, or downstream of the gene body in a non-conserved region) for 8kb knock-in.
2. Do you know the copy number of the target gene in the cell line? Are you aiming for homozygous or heterozygous clone?
3. You might want to ensure that the 5 SNPs are actually on the same allele after targeting. Long PCR + sequencing is one potential way to do it - but confirming this could be challenging.