I need to do in vitro digestion, and I would like to increase the efficiency. Now I'm using Cas9 from NEB and follow their protocol:
https://www.neb.com/protocols/2014/05/01/in-vitro-digestion-of-dna-with-cas9-nuclease-s-pyogenes-m0386
I have created a test assay - PCR amplicon and a gRNA targeting it. And I would like to get the efficiency as high as possible.
The standard protocol gives ~40%. Increased reaction time (up to 16h) improves it to 60%.
I wonder if someone has already tried the other ways of optimization.
Hello
Check te molar ratio of Cas9 Nuclease:sgRNA:target site, should be 10:10:1 and check the integrity of the sgRNA. The good quality of sgRNA and optimal aligment to target site is important for efficiency.
Hi!
I have checked the molar ratio and the integrity of sgRNA.
As for the alignment to target - this is the best sgRNA for 600bp genomic region. The score of http://crispr.mit.edu/ is 98/100 for this sgRNA.
On the neb web site they say that the molar ratio should be at_least 10:10:1. Have you tried to increase it?
Hi
I used higher ratio (15:15:1) in few difficult cases but max efficiency was ~80%, but many times I also used 5:5:1 and it worked.
I found information that that including a nonspecific protein such as insulin prevents the nucleosomes from falling apart and getting lost to surfaces during binding and cleavage assays Additionally, inclusion of insulin reduces nonspecific protein-protein interactions and aggregation. Maybe it will help you.
we need to use very concentrated cas9 to make 15:15:1 and not increasing the cas9 ratio more than %10 in the reaction.
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