2.3 Embryo microinjection
Fertilized eggs were collected (within 2 hours after laying) and surface
sterilized by soaking the eggs in 1% sodium hypochlorite solution for 1
minute and washed with distilled water for three times. The eggs were
arranged on double-sided adhesive tape on a microscope slide. Two sgRNAs
of target loci of GOBP1 and GOBP2 with the TrueCutTMCas9
Protein v2 (Thermo Fisher, Shanghai, China) were mixed, using the
RNase-free water adjust the final concentration of sgRNA (200 ng/μL)
with Cas9 protein (100 ng/μL). Each embryo was injected with
approximately 1 nL of the mix solution containing sgRNA and Cas9
protein. The injected eggs were first placed on the ice maintained for
20 min then incubated at 27 ± 1 ℃ until hatching.