Crimson Pixie
posted over 4 years ago

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.

Orchid Gnomeover 4 years ago

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.

Crimson Pixieover 4 years ago

Thank you for your answer,

We are actually interested in an intergenic region and would like homozygous clones.

We have actually done both PCR + direct sequencing as well as cloning and sequencing of the region to check for the SNPs and found to be heterozygous in the cell lines that we are working with. So, technically, even if we get a heterozygous clone from CRISPR, we might end up with a homozygous clone by chance. Because the cells are heterozygous to begin with.

So the question is, Is it possible to "replace" a fragment of 8kb?

Orchid Gnomeover 4 years ago

Short answer is yes, it is feasible to replace 8kb. Even before CRISPR, people replace hundreds of kb sequences in ES cells routinely. You might need selection marker to increase the efficiency for certain cell lines.

If you are starting with heterozygous cell, the genotyping will be easy too - just sequence every SNP and confirm it is homozygous. You might run into clones with partial recombination, as homologous sequences in between SNPs are likely to be big enough to serve as homology arms.

I would be very careful with the "heterozygous" status of the starting cell line though. Unless you did long PCR spanning full 8kb, TA cloned the fragment, and sequenced single clones, it is hard to tell whether all SNPs are actually from the same allele.

Login or Signup to leave a comment