Why is CRISPR-Cas9 easier to design for many targets than zinc finger nucleases?

CRISPR-Cas9 is easier to design for various targets than zinc finger nucleases (ZFNs) primarily because it utilizes RNA-DNA binding rather than protein-DNA binding to guide nuclease activity.1

Designing ZFNs requires the complex engineering of fusion proteins that recognize and bind to specific DNA sequences. To achieve a double-stranded break, researchers must engineer two separate ZFN fusion proteins—one for each side of the target site—which then must dimerize to become active.1

In contrast, the CRISPR-Cas9 system relies on the stability of RNA-DNA heteroduplexes. Because designing an RNA sequence to bind to a specific target DNA sequence requires only knowledge of simple Watson-Crick base-pairing rules, the process is significantly more straightforward and versatile. This simplicity allows CRISPR-Cas9 to be applied to a much broader range of target sequences compared to the labor-intensive protein-based designs required for ZFNs.12