Generation of Conditional Knockout Mice by Sequential Insertion of Two loxP Sites In Cis Using CRISPR/Cas9 and Single-Stranded DNA Oligonucleotides.

Conditional knockout (cKO) mice are extremely valuable for biomedical research because they enable detailed analyses of gene functions in a tissue- or temporally-specific fashion. The conventional method for generating cKO mice is time consuming and labor intensive, which involves making a large gene-targeting construct, transfecting and screening many embryonic stem (ES) cell clones, injecting positive ES clones into blastocysts to produce chimeric mice, and breeding the chimeras to transmit the targeted gene through the germline. Recently developed CRISPR technology has revolutionized the way genetically engineered organisms are created. Knockout and knockin mice can now be made by directly injecting zygotes with Cas9, sgRNA, and donor DNA. In theory, cKO mice can be generated by simultaneously inserting two loxP sites using two sgRNAs and two oligonucleotides as donors, but in practice the probability of obtaining cKO mice in one step is still very low, partly because the efficiency of oligo-mediated knockin is much lower than non-homologous end joining (NHEJ) and partly because co-cutting juxtaposed sites in one allele at the same time often leads to the deletion of the entire fragment between the two cutting sites. Therefore, many laboratories prefer to insert the two loxP sites sequentially, i.e., generating mice with one loxP first and then use embryos collected from these mice to insert the second loxP site. In this chapter, we describe our procedures and timeline using this sequential method to make a Six6 cKO mouse line as a demonstration of its feasibility.