CRISPR/Cas9 system is a system normally used by some microorganisms to repair DNA defects in their genome. This system is now used as an efficient tool for genome engineering. I am currently trying to clone an oligo (annealed from two designed primers) using the kit, which include the vector to be used. However, I am only getting a very little amount of clones. I am wondering if maybe the efficiency of the system or the kit is normally very low or is there a problem with my primers? But I don't think the primer is the problem because I tried to clone a different gene using different primers and I am getting the same kind of results. Can anyone with experience with CRISPR/Cas9 system assist with some advice please? Please note the kit I am using is from Life Technologies [GeneArt CRISPR Nuclease Vector Kit]
Dr. Feng Zhang's lab has a protocol well established for the most commonly used crispr backbones. Maybe you can try setting up your cloning reaction in a similar manner. Here's the link for the protocol
http://www.genome-engineering.org/crispr/wp-content/uploads/2014/02/CRISPR-Reagent-Description-Rev20140220.pdf
A lot of thngs can cause the problem, first did you check your oligos for secondary structures that might cause problems, purity of oligos is also important and the annealing conditions. Using phosphorylated oligos can improve cloning efficiency, sometimes dramatically. These are actually general considerations for oligonucleotides cloning.
Dr. Feng Zhang's lab has a protocol well established for the most commonly used crispr backbones. Maybe you can try setting up your cloning reaction in a similar manner. Here's the link for the protocol
http://www.genome-engineering.org/crispr/wp-content/uploads/2014/02/CRISPR-Reagent-Description-Rev20140220.pdf
Using Zhang's lab protocol and also one of their CRISPR-Cas9 plasmid obtained from Addgene we got plenty of clones, even without phosphorylation of the oligos. Could be that you have a problem with the competent cells?
Dear Dacquin,
Please find the below link for all the protocols and fullfilized details about CRISPR/Cas system in a single ebook from Addgene company
http://info.addgene.org/download-addgenes-ebook-crispr-101-2nd-edition
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