CLICK: One-step generation of conditional knockout mice

CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxPflanked (floxed) alleles remain elusive. We show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats.