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How do zinc finger nucleases create double-stranded breaks in DNA?
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genetic engineering
The generation of vaccines relies heavily on techniques in genetic engineering.
Zinc finger nucleases (ZFNs) create double-stranded breaks in DNA through a precise, engineered process involving fusion proteins. Each ZFN is composed of two primary components:
- DNA-binding domains: These domains are engineered to recognize and bind to specific three- to four-base-pair-long sequences. By fusing multiple domains in tandem, researchers can target longer, specific DNA sequences.
- Fok1 nuclease subunit: Each DNA-binding domain is fused to a subunit of the bacterial nuclease Fok1.
To facilitate a double-stranded cut, two distinct ZFN fusion proteins must be engineered—one designed to bind to each side of the target site on opposite strands of the DNA. When both ZFNs successfully bind to their respective target sequences, the Fok1 subunits are brought into close proximity. This allows them to bind to each other and form an active dimer. Once dimerized, the Fok1 nuclease is capable of cutting the target DNA on both strands, resulting in a double-stranded break at the desired location.1