Efficient knock-in method enabling lineage tracing in zebrafish

Abstract

Here, we devised a cloning-free 3′ knock-in strategy for zebrafish using PCR amplified dsDNA donors that avoids disrupting the targeted genes. The dsDNA donors carry genetic cassettes coding for fluorescent proteins and Cre recombinase in frame with the endogenous gene but separated from it by self-cleavable peptides. Primers with 5′ AmC6 end-protections generated PCR amplicons with increased integration efficiency that were coinjected with preassembled Cas9/gRNA ribonucleoprotein complexes for early integration. We targeted four genetic loci (krt92,nkx6.1,krt4, andid2a) and generated 10 knock-in lines, which function as reporters for the endogenous gene expression. The knocked-in iCre or CreERT2 lines were used for lineage tracing, which suggested thatnkx6.1+cells are multipotent pancreatic progenitors that gradually restrict to the bipotent duct, whereasid2a+cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. In addition, the hepaticid2a+duct show progenitor properties upon extreme hepatocyte loss. Thus, we present an efficient and straightforward knock-in technique with widespread use for cellular labelling and lineage tracing.