I managed to make a deletion using the plasmids from the Friedland et al. and only changing the sequence for sgRNA. Even though with this first try, I obtained multiple alleles, for the next 4 genes I tried I didn’t get any positive results. What is your experience with the CRISPR so far and which method are you using?
Maybe with a good discussion here, we can get an idea on which methods work best?
Hi, a few quick questions:
-how are you screening for deletions? (phenotype, PCR with either restriction digest or mismatch cutting enzyme?)
-have you tried multiple sgRNAs for the next four genes?
-how many animals have you screened for each gene you are targeting?
The rules of what makes a good sgRNA are not clear yet, though are likely being explored. It might be that the sgRNAs that you chose are intrinsically not great or that the genes you are targeting are difficult to cut. Indeed, Friedland and others have seen huge variations in targeting efficiency (0.5-80%) depending on the gene.
Another idea could be to use paired sgRNAs spaced apart. This might either increase your probability of getting a deletion or even create a larger deletion.
I am using T7E1 enzyme for cutting heteroduplexes. It worked great in the gene I made several alleles. I am screening usually between 50 - 150 F1s depending
on the availability of marker positive animals. Though based on Bono Lab paper, marker negative animals should give positive results as well. That’s how it works in Mos too.
I always design 3 sgRNAs for every gene targeted and inject together.
It is possible that all the sgRNAs were the bad ones and/ or the genes are the hard to get ones. I was wondering in general how are the others doing?
Anybody experienced that one cas9 plasmid was better than the other or any differences between different U6 promoters used?
Yeah, it seems that it might be worth screening marker negative animals as well. In Ding Xue’s recent paper on using oligos to knock sequence in, he had better luck with marker negative animals (though his problem was that the marker positive animals) had indels instead of knockins. I don’t think that anyone has compared any of the U6 driven sgRNAs head-to-head. There seem to be differences based on the papers, but it’s hard to compare. I haven’t heard of one Cas9 being better though. Maybe also try the RNA injection method (or protein-RNA if you can get your hands on it)? Maybe that would give you a higher dose of Cas9 and sgRNA and really drive indel formation?