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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 is our current protocol for generating Cas12a crRNAs for injection in zebrafish.
Note - we no longer use the "GC" swap for the crRNA backbone. Across many more targets that we have tested in zebrafish since the NAR paper, we find that the extended "dr"-crRNA without GC works best. This is reflected in the PDF here. 

Generation of crRNA templates, crRNA, and injection (updated 8/28/20): PDF.


We find that our purified recombinant protein made in-house works best.  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.