Well begun is half done. But if you are making a CRISPR knock-out mouse, it is hard to tell early enough whether you’ve begun well. Thankfully, a talented team of researchers in Japan led by Takayuki Sakurai have now optimized an assay that helps you do just that – predict if you are on the right path to generating a knock-out mouse, as early as 3 days into the procedure.
CRISPR genome editing is a relatively speedy way of generating knockout mice today. That said, you must still go through the non-trivial process of microinjecting zygotes with a guideRNA-Cas9 mRNA mix followed by embryo transfer into pseudo-pregnant female mice. And then, it is only when pups are born, some 20 days later, that you find out if your attempt at generating a knock-out mouse worked indeed.
If not, you’re right back to where you started, designing a different guide-RNA to achieve that knockdown.
Sakurai and colleagues optimized an assay that saves researchers this heart-ache: assaying for the presence of CRISPR-induced indel mutations in single cultured blastocysts, just 3 days post-injection.
Their new protocol involves:
- Following injection of the guideRNA-Cas9 mRNA mix, in-vitro culturing of the zygotes to the blastocyst stage
- PCR amplifying the region of interest from crude DNA preparations obtained by lysing single blastocysts
- Using the T7 endonuclease I digestion of PCR products to detect indel mutations. The digestion products can be further sequenced to discern the nature of an indel mutation.
What about off-target effects, you ask? The team also performed reliable whole genome amplification of crude DNA preparations from single blastocysts. This boosts the available starting material, which could be used for analyzing off-target effects.
Go check out the full details at BMC Biotechnology July, 2014.