Efficient knock-in method enabling lineage tracing in zebrafish

The CRISPR-Cas9 system aids generation of knock-in zebrafish lines, but it has been hard to integrate large constructs and avoid disrupting the targeted genes. Here we devised a 3’ knock-in strategy of PCR-amplified dsDNA, which coded for fluorescence proteins and Cre recombinase in frame with the endogenous gene but separated from each other by self-cleavable peptides. Primers with 5’ AmC6 end-protections generated improved PCR amplicons harboring either short or long homologous arms, which were co-injected with pre-assembled Cas9/gRNA ribonucleoprotein complexes for early integration. We targeted four genetic loci (krt92,nkx6.1, krt4,andid2a) and generated ten knock-in lines, which function as reporters for the endogenous gene expression. The knocked-in iCre or CreERT2 were used for lineage tracing, which suggestednkx6.1⁺cells are multipotent pancreatic progenitors that gradually restrict to bipotent duct; whileid2a⁺cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. Additionally, hepaticid2a⁺duct show progenitor properties upon extreme hepatocyte loss. Thus, we present an efficient knock-in technique with widespread use for both cellular labelling and lineage tracing.