Identification Of Sites For Internal Insertions Into Cas9

Patent Status

Brief Description

The ability to add a protein domain of new function is a standard molecular biology technique, and usually the domain is fused to a protein terminus. The CRISPR-associated protein Cas9 already has widespread utility for genome engineering, yet adding protein domains would increase precision and specificity. Both protein termini of Cas9, however, are close to each other and in a small defined region, which limits the effectiveness of standard fusion approaches. Therefore, insertion sites within Cas9 that will not disrupt Cas9 function are needed.

Researchers at UC Berkeley have identified over 150 such sites. In proof-of-concept experiments, a PDZ protein interaction domain has been intercalated and increased functionality without decreasing Cas9 nuclease activity. In further experiments, the internal insertion sites have been used to alter Cas9 activity in an allosteric manner, effectively creating tunable Cas9.

Suggested uses

- Biosensors that control gene expression in response to inputs
- Therapeutics
- Basic research tool

Advantages

- Spatiotemporal control over Cas9 binding/cleavage
- Higher specificity of Cas9 activity
- Compatible with a wide variety of known protein domains

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