Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 gene editing relies on protospacer-adjacent motif (PAM) recognition.
• This PAM requirement restricts the available target DNA sequences for genome manipulation.
• Existing CRISPR-Cas9 systems, like *Streptococcus pyogenes* Cas9 (SpCas9), are limited by their specific PAM sequences (e.g., NGG).

Purpose of the Study:

• To engineer SpCas9 variants that eliminate or reduce the dependence on the canonical NGG PAM.
• To expand the targeting range of CRISPR-Cas9 for broader genome editing applications.
• To develop novel tools for generating previously inaccessible genetic variants, including disease-relevant ones.

Main Methods:

• Protein engineering of SpCas9 to create variants with altered PAM specificities.
• Development of SpG variant targeting NGN PAMs.
• Further optimization to create the SpRY variant, targeting NRN and NYN PAMs.
• Assays to evaluate nuclease and base-editor activities of SpG and SpRY in human cells.

Main Results:

• Engineered SpG variant successfully targets NGN PAM sequences.
• Developed SpRY variant exhibits broad PAM targeting, including NRN and NYN motifs.
• SpRY showed robust activity on NRN PAM sites and substantial activity on NYN PAM sites in human cells.
• SpG and SpRY enabled the generation of disease-relevant genetic variants not achievable with wild-type SpCas9.

Conclusions:

• SpG and SpRY significantly expand the targeting scope of CRISPR-Cas9 technology.
• These engineered variants overcome PAM limitations, enabling high-resolution genome editing.
• The developed tools are valuable for diverse applications, including the study of genetic diseases.