RT Journal Article SR Electronic T1 Efficient knock-in method enabling lineage tracing in zebrafish JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e202301944 DO 10.26508/lsa.202301944 VO 6 IS 5 A1 Jiarui Mi A1 Olov Andersson YR 2023 UL https://www.life-science-alliance.org/content/6/5/e202301944.abstract AB 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, and id2a) 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 that nkx6.1+ cells are multipotent pancreatic progenitors that gradually restrict to the bipotent duct, whereas id2a+ cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. In addition, the hepatic id2a+ 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.