Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas gene editing systems offer precise DNA targeting but struggle with genetic variations like single nucleotide polymorphisms (SNPs).
• The rigidity of DNA sequence recognition in CRISPR systems limits their ability to target multiple gene variants with a single guide RNA.

Purpose of the Study:

• To investigate the potential of incorporating universal bases into guide RNAs for CRISPR/Cas systems.
• To enhance the flexibility of CRISPR/Cas systems for simultaneous targeting of polymorphic DNA sequences.
• To expand the targeting capabilities of CRISPR/Cas systems beyond canonical spacer sequences.

Main Methods:

• Demonstrated Cas9 tolerance to universal bases within individual guide RNAs.
• Assessed specificity changes at the site of universal base incorporation.
• Applied the technology to target multiple human SNPs and design Cas12a/Cpf1-based DETECTR probes for HIV protease gene variants.

Main Results:

• Cas9 successfully tolerated universal bases in guide RNAs, enabling simultaneous targeting of polymorphic sequences.
• Specificity remained preserved at non-universal base incorporation sites, with selective degeneracy only at the universal base position.
• Successfully targeted multiple human SNPs and identified evolved HIV protease gene variants using modified CRISPR systems.

Conclusions:

• Incorporating universal bases into guide RNAs significantly enhances the versatility of CRISPR/Cas gene editing.
• This approach allows for the simultaneous targeting of diverse genetic variants, including SNPs and evolved pathogen sequences.
• The findings introduce a novel application for universal bases in expanding the scope of CRISPR/Cas-based diagnostics and therapeutics.