Problem	Solution
No commercial Cas9 protein for sgRNA test.	In vitro transcription and purification of Cas9 mRNA and use it to test sgRNA efficiency.
Low maternal mutant frequency.	Prescreen Tg(zpc:zcas9) by injecting bmp2b sgRNA into the embryos and select the fish with injected embryos displaying a high ratio of dorsalized phenotypes.
Failure in amplification of the coding sequence.	Use nested PCR.