Engineered prime editors with PAM flexibility.

Area of Science:

• Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• Prime editors offer precise genome editing without double-strand breaks or donor DNA.
• Conventional prime editors using Streptococcus pyogenes Cas9 (spCas9) are limited by Protospacer Adjacent Motif (PAM) recognition.
• Expanding the target scope of prime editing is crucial for broader biological research applications.

Purpose of the Study:

• To engineer prime editors with expanded target scopes by utilizing PAM-flexible Cas9 variants.
• To demonstrate the efficacy of engineered prime editors in generating diverse mutations.
• To overcome the limitations of conventional prime editors in targeting specific mutations, such as BRAF V600E.

Main Methods:

• Engineering of prime editors incorporating various PAM-flexible Cas9 variants.
• Testing prime editing activity and mutation generation in HEK293T cells.
• Introduction of specific mutations, including BRAF V600E, using engineered prime editors.

Main Results:

• Successfully generated over 50 types of mutations using engineered prime editors.
• Achieved up to 51.7% prime-editing activity in HEK293T cells.
• Successfully introduced the BRAF V600E mutation, which is not achievable with conventional prime editors.

Conclusions:

• Engineered prime editors utilizing PAM-flexible Cas9 variants significantly broaden the target range of prime editing technology.
• These advancements enhance the applicability of CRISPR-based prime editing for diverse biological research.
• The developed prime editor variants enable precise introduction of mutations previously inaccessible to conventional systems.