Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function.However,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported.Here we first developed a strat-egy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system.We demonstrated the feasibility of this strategy at sox10 and isI1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluores-cent reporter,allowing generation of genetic mosaics for lineage tracing.We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alle-les,both tagged with two different fluorescent reporters.By introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morpho-logically visible phenotypes.We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus.Furthermore,we eliminated the undesir-able bacterial backbone in the donor using minicircle DNA technology.Our system could easily be expanded for other applications or to other organisms,and cou-pling fluorescent labeling of gene expression and con-ditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.