Mint Vampire
posted over 4 years ago

Unwanted deletions after CRISPR/Cas9...how big can they become ?

When working with CRISPR approaches all kind of mutations can occur through NHEJ. Most frequently I and others see small (1-5nt) deletions, less frequently insertions, sometimes lager deletions.

The biggest deltion I have found recently on a mouse ES cell projest is 350 nt. In a recent report from Alan Bradley (https://www.nature.com/articles/nbt.4192#s4) a much bigger set of deletions have been reported even a 9kb deletion through a singe sgRNA !

DO people here see also large unwanted deletions in their projects ? It might shed another light on one of my current projects.

Sienna Faunover 4 years ago

We have had a few occasions in which we mated CRISPR founder animals and got PCR genotypes in the offspring that didn't make sense, suggesting there might be larger deletion alleles present that were not detected due to deletion of one or both PCR primer sites. We subsequently re-genotyped with primers spaced further apart and detected larger deletions present in the founders that were not detectable with our first primer sets. Most of these have been on the order of a few hundred bp, but it is possible there were larger ones that went undetected. You only see what your assay is designed to look for.

Green Gremlinover 4 years ago

See them all the time in mice. Sometimes it looks as though we have a homozygote mutation in a founder for example, but when bred forward not every pup gets this allele, indicating the presence of an unidentified allele in the founder. Not a problem for us, we breed forward the alleles we want, but if people are considering engineering cultured cells where segregation of alleles is not possible these outcomes have to understood, identified and carefully considered.

Mint Vampireover 4 years ago

Indeed for breeding it might not be a problem, but in this case the ES cells are to be used for CHIP experiments. Just today I found out that my homozygous line was in fact a hemizygous one: One allel had the desired Ab tag, the other Wt allele did not expres the Wt protein, due to a 2.4 Kb deletion. My Southern Blot probe as wel as my first set of Long Range PCR primers fell into this deletion, making me draw the false conclusion.

Lemon Golemover 4 years ago

in cases like that... how about including an allele counting real-time PCR assay as was used in the papers from the Regeneron groups on using BACs as targeting vectors? Our own experience with them is that they're not always fully conclusive on their own and sometimes tricky to set up, but in combination with long-range PCR and Southern blot I might be all you need for the final tests... It should be able to pick out these sort of issues with large deletions...

Bronze Impover 4 years ago

Another paper from our lab supporting the large deletion in mouse zygote injection and in ES cells https://www.nature.com/articles/s41586-018-0380-z

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