Figure 8

Genome editing operations using targetrons and recombinases. Cre/lox is the recombinase system used in this example. (A) Inserting exogenous DNA (recombinase-mediated cassette exchange). Two lox sites having incompatible linker regions and differing arm mutations (for example, lox71 and lox66) are delivered to the genome using an intron. The sequence to be inserted is then delivered between lox sites identical to those in the genome except having opposite arm mutations. The formation of non-functional lox sites (lox72) makes the process irreversible. (B) Procedure for deleting genomic sequences. After delivery of lox sites (lox71 and lox66) on targetrons, Cre-mediated recombination then deletes the intervening region, leaving a non-functional lox site (lox72) behind. (C) Procedure for inverting genomic sequences. The procedure is the same as in panel B, except the lox sites have opposing orientations. (D) Procedure for one-step cut-and-paste after using introns to position lox sites (two lox71 sites and one lox66 site) as shown. The first (reversible) step is Cre-mediated deletion, followed by Cre-mediated reinsertion at the target site that is made irreversible by the formation of a non-functional lox site (lox72).