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Using Cas12a in zebrafish

Cas12a (also referred to as Cpf1) is a recently described CRISPR-associated (Cas) protein that can efficiently introduce targeted DNA breaks in mammalian cells and zebrafish embryos. In collaboration with the Wolfe Lab, we have recently identified optimized variants of Cas12a and its crRNA and applied them in zebrafish, where they are highly active. Below are protocols for generating optimized Cas12a crRNA variants.

Generation of crRNA templates, in vitro crRNA, and injection: PDF.

We use purified recombinant protein made by the Wolfe Lab. To generate your own, you can obtain the pET plasmid from Addgene

Alternatively, you can purchase LbCas12a from New England Biolabs. We have confirmed that this Cas12a works in zebrafish, although it is not as active as our version, likely due to differences in the orientation of nuclear localization sites. 

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