Recombinant Expression and Application of mSaCas9 Protein Based on Codon-optimization
[Objective]Using SaCas9 from Staphylococcus aureus to study gene editing proteins that can replace commercial Cas9,to provide a reference for the production of proteases suitable for gene editing in fish.[Method]Through optimizing SaCas9(named mSaCas9)with the codon preference of medaka(Oryzias latipes),cloning and constructing recombinant plasmid pET28a-mSaCas9,and obtaining prokaryotic expression of mSaCas9 recombinant protein by using Escherichia coli BL21(DE3),the activity of the purified protein was verified by in vitro enzyme digestion;the applicability of the mSaCas9-RNP delivery method was verified by microinjection using the purified protein.[Result]The recombinant plasmid pET28a-mSaCas9 was successfully constructed,and its recombinant protein had a molecular mass of 130 ku;2.5 mg of mSaCas9 protein was purified in 1 L of medium with a purity of 95%;in vitro enzyme digestion showed that the PCR products of tyr and oca2 genes could be edited at a final concentration of 30 ng/μL,which demonstrated that the purified protein possessed editing activity in vitro.The mSaCas9 protein and oca2-gRNA of melanin synthesis-related gene were injected into medaka embryos,and the eyes of the embryos showed pigmentation loss,which indicated that mSaCas9-RNP could be applied to medaka genome editing.[Conclusion]The active Cas9 protein with smaller molecular mass was obtained by in vitro expression and purification,and the effectiveness of gene editing at the individual level by delivering SaCas9 protein and gRNA,i.e.,SaCas9-RNP(SaCas9 ribonucleoprotein),was demonstrated in fish.
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