Synthetic Biology Parts, Devices and Applications

40 3 Site-Directed Genome Modification with Engineered Zinc Finger Proteins

DNA

Fok 1

Fok 1

PCR amplification of targeted site

Heating and reannealing of strands

Digest with surveyor nuclease

Gel electrophoresis

Control Targeted

Cut

Uncut

Cleaved

Cleaved

Mismatched duplex DNA

Mismatched duplex DNA

Figure 3.2 A nuclease assay for detecting gene targeting. ZFNs are used to create a targeted

DNA DSB. PCR is used to amplify the targeted sites and the DNA is heated and cooled to

reanneal the strands, creating mismatched heteroduplexes of DNA. The Surveyor nuclease

cleaves the heteroduplexes only at the sites of mismatched DNA, leaving the homodimers

unmodified. Gel electrophoresis can then be used to observe and quantitate the efficiency of

gene targeting using ZFNs at the genomic level.

3.4 Validating Zinc Finger Nuclease-Induced Genome

Alteration and Specificity

Methods have been developed for monitoring for endogenous gene modifica-

tion. One such assay evaluates for DNA DSB repair via using the Surveyor nucle-

ase [35]. This assay involves three steps: (i) polymerase chain reaction (PCR)

amplification of the region of interest and annealing of the strands to form

homoduplexes (no mismatches) and heteroduplexes (containing mismatches),

(ii) cleavage of the mismatched heteroduplexes by the Surveyor nuclease, and

(iii) fragment size evaluation to determine if mismatched DNA was present [61].

By only cleaving annealed complexes containing both the mutated and wild-type

DNA after amplification, the Surveyor nuclease can used to estimate the level of

mutagenesis mediated by the ZFNs (Figure 3.2).