Synthetic Biology Parts, Devices and Applications

34 3 Site-Directed Genome Modification with Engineered Zinc Finger Proteins

use of engineered ZFPs called zinc finger nucleases for site-directed genomic

modification through targeted DNA cleavage. When a targeted double-stranded

DNA break is engineered, endogenous repair subsumes via either homologous

recombination (HR) or nonhomologous end joining (NHEJ) [3] (Figure 3.1).

3.1.2 Homologous Recombination

HR is a process of exchanging shared DNA sequences between sister chromatids.

HR naturally occurs in a diverse range of organisms, from bacteria to humans.

The HR process has two major purposes: (i) the protection of somatic genomes

through DSB repair to prevent mutations that could result in cell death or cancer

and (ii) to increase the genetic diversity of the next generation through recombi-

nation of the parental chromosomes in each gamete during meiosis. HR has been

DNA

Deletion

Gene disruption Gene correction

Targeted gene addition

NHEJ HR

Fok 1

Fok 1

Figure 3.1 Targeted genomic modification using zinc finger nucleases (ZFNs). A pair of ZFPs

fused to the FokI nuclease domain is designed to target opposite strands of DNA. When

dimerization of the FokI domain occurs following ZF binding, a double-strand break (DSB) is

created in the DNA (shown by lightning bolt). The cell chooses to use either the

nonhomologous end joining (NHEJ) or homologous recombination (HR) pathway to repair the

DSB. NHEJ can be used for gene disruption via targeted mutagenesis using one pair of ZFNs or

deletions/inversions with two pairs of ZFNs. If gene correction via HR is desired, a homologous

template sequence is provided that will be used by the cellular HR machinery replace the

endogenous sequence near the DSB. Alternatively, targeted gene addition at or near the site

of the targeted DNA cleavage can be achieved by flanking the sequence to be inserted with

homologous arms.