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Published on: June 16, 2017
Peptide Nucleic Acid-Mediated Regulation of CRISPR-Cas9 Specificity
Kelly E W Carufe1,2, Nicholas G Economos1,2,3, Peter M Glazer1,2
1Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut, USA.
Abstract:
Although CRISPR-Cas9 gene therapies have proven to be a powerful tool across many applications, improvements are necessary to increase the specificity of this technology. Cas9 cutting in off-target sites remains an issue that limits CRISPR's application in human-based therapies. Treatment of autosomal dominant diseases also remains a challenge when mutant alleles differ from the wild-type sequence by only one base pair. Here, we utilize synthetic peptide nucleic acids (PNAs) that bind selected spacer sequences in the guide RNA (gRNA) to increase Cas9 specificity up to 10-fold. We interrogate variations in PNA length, binding position, and degree of homology with the gRNA. Our findings reveal that PNAs bound in the region distal to the protospacer adjacent motif (PAM) site effectively enhance specificity in both on-target/off-target and allele-specific scenarios. In addition, we demonstrate that introducing deliberate mismatches between PNAs bound in the PAM-proximal region of the gRNA can modulate Cas9 activity in an allele-specific manner. These advancements hold promise for addressing current limitations and expanding the therapeutic potential of CRISPR technology.
Insights
Synthetic peptide nucleic acids (PNAs) enhance CRISPR-Cas9 gene editing specificity up to 10-fold by targeting guide RNA. This improves precision for human therapies and allele-specific disease treatments.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetic Engineering
Background:
- CRISPR-Cas9 gene therapy offers broad applications but requires enhanced specificity.
- Off-target mutations and challenges in treating single-nucleotide difference diseases limit current CRISPR-Cas9 technology.
- Improving Cas9 specificity is crucial for advancing human-based gene therapies.
Purpose of the Study:
- To develop a method for increasing CRISPR-Cas9 specificity using synthetic peptide nucleic acids (PNAs).
- To evaluate the impact of PNA modifications (length, binding site, homology) on Cas9 specificity.
- To explore allele-specific gene editing capabilities enabled by PNA-guided Cas9.
Main Methods:
- Synthetic peptide nucleic acids (PNAs) were designed to bind specific sequences on guide RNA (gRNA).
- Variations in PNA length, binding position relative to the protospacer adjacent motif (PAM), and homology were tested.
- Cas9 specificity was assessed in both on-target/off-target and allele-specific editing contexts.
Main Results:
- PNAs significantly increased Cas9 specificity, achieving up to 10-fold enhancement.
- PNAs binding in the gRNA region distal to the PAM site effectively improved specificity.
- PNAs in the PAM-proximal region with deliberate mismatches allowed for allele-specific modulation of Cas9 activity.
Conclusions:
- Peptide nucleic acids represent a promising strategy to enhance CRISPR-Cas9 specificity and precision.
- This approach addresses limitations in current gene editing technology, particularly for therapeutic applications.
- The findings pave the way for more accurate and effective CRISPR-based treatments for genetic diseases.
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