Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes is a key RNA-guided DNA endonuclease for genome modification.
• Understanding the structural limitations for engineering Cas9 is crucial for expanding its applications.

Purpose of the Study:

• To experimentally profile Cas9 structure and identify sites amenable to domain insertions without compromising function.
• To demonstrate the creation of novel Cas9 functionalities through targeted domain insertions.

Main Methods:

• Randomized insertional mutagenesis was employed to profile Cas9 structure.
• Identification of structural 'hotspots' that tolerate PDZ domain insertions.
• Insertion of orthogonal domains and the estrogen receptor-α ligand-binding domain into identified sites.

Main Results:

• Specific structural hotspots in Cas9 were identified that tolerate domain insertions without affecting DNA binding and cleavage.
• Orthogonal domains and domain combinations could be inserted with minimal functional impact.
• An allosterically regulated Cas9 was successfully constructed via domain insertion, exhibiting ligand-dependent activation in both prokaryotic and eukaryotic cells.

Conclusions:

• Domain insertion profiling is an effective strategy for rapidly generating new Cas9 functionalities.
• The identified sites provide a basis for future engineering of Cas9, enabling versatile, inducible, and reversible genome editing systems.