I've performed microinjections to create a tag-insertion inside my gene of interest by CRISPR/Cas9. A quick PCR shows that a bunch of the pups are positive. However, all are heterozygotes.
I'm looking for recommendations for:
1. a cloning-free screening method to determine if the insertion is correct by sequencing.
2. a method to screen for off-target effects.
1) you could use the surrounding sequence for a primer design and another from within the heterologous dna segment. if the PCR is positive you will know it is in the right position. This does not mean however that it might not be inserted also somewhere else !! It is widely known that the crisp/cas9 guidance system is not that accurate under all conditions.
2) With off target you mean random insertions ??. Yo can look at this by southern analysis.
good luck,
marten smidt
Hi Marten, thank you for your answer.
1) I've performed such kind of PCR and I know I've got several positive mice. I also know that these animals are all heterozygous. I would like to perform sequencing to check whether the tag is correctly inserted and inframe with the gene. I was wondering if there is a sequencing method which can delineate overlapping peaks from a convoluted spectrum using genomic material as template. Meanwhile I have found this paper which proposes an algorithm that can do this: http://www.nature.com/articles/srep28973
Alternatively, I can just send in the PCR product for sequencing. This will require gel extraction and purification. Since I have a bunch of samples and I'm expecting more with upcoming microinjections, I was looking for a more efficient method.
2) Southern analysis is a good way to go. I was thinking either that or FISH.
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