Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 genome editing relies on recognizing specific DNA sequences called protospacer adjacent motifs (PAMs).
• The widely used Streptococcus pyogenes Cas9 (SpCas9) is limited to NGG PAM sequences.
• Existing Cas9 variants offer limited PAM flexibility for mammalian cell applications.

Purpose of the Study:

• To engineer a Cas9 variant with an expanded PAM recognition profile.
• To assess the functionality and specificity of the engineered variant in human cells.
• To broaden the targeting scope of CRISPR-Cas9 technology.

Main Methods:

• Phage-assisted continuous evolution (PACE) was employed to evolve an expanded PAM SpCas9 variant (xCas9).
• Functional assessments included targeted transcriptional activation, gene disruption, and base editing in human cells.
• Genome-wide off-target activity was evaluated to determine DNA specificity.

Main Results:

• The evolved xCas9 variant recognizes a broad range of PAM sequences, including NG, GAA, and GAT.
• xCas9 demonstrates the broadest PAM compatibility reported for Cas9 proteins active in mammalian cells.
• xCas9 exhibits significantly higher DNA specificity and lower off-target activity compared to SpCas9.

Conclusions:

• The engineered xCas9 expands the DNA targeting capabilities of CRISPR systems.
• This variant supports diverse CRISPR applications in human cells with enhanced precision.
• Expanded PAM compatibility does not necessitate a trade-off with Cas9 editing efficiency or DNA specificity.