Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene functions. Han et al. developed a high-efficient bidirectional knockin strategy called Bi-FoRe to simultaneously generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters, through NHEJmediated bacterial backbone-free targeted insertion facilitated by CRISPR/Cas system in zebrafish. Conditional knockout and conditional gene restoration could be achieved through Cre-induced inversion of positive and negative alleles, respectively, coupled with switch of fluorescent reporters, allowing generation of genetic mosaics for lineage tracing. Furthermore, differential fluorescent labeling of positive and negative alleles enables early, simple and efficient discrimination of individual live embryos bearing different genotypes, prior to emergence of morphological phenotypes and in large quantities.