Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing

Area of Science:

• Biochemistry
• Molecular Biology
• Genetic Engineering

Background:

• RNA-based therapeutics utilize chemical modifications to enhance stability and reduce immune responses.
• CRISPR-Cas9 gene editing relies on guide RNAs (crRNA and tracrRNA) for DNA targeting.
• Optimizing guide RNA chemical modifications is crucial for advancing CRISPR-Cas9 therapeutic potential.

Purpose of the Study:

• To investigate the impact of chemical modifications across all positions of crRNA and tracrRNA.
• To identify modified guide RNAs that exhibit increased potency and stability compared to unmodified versions.
• To develop fully chemically modified guide RNAs functional in human cells for CRISPR-Cas9 applications.

Main Methods:

• Systematic chemical modification of guide RNAs (crRNA and tracrRNA) at all nucleotide positions.
• In vitro and in vivo functional assays to assess the potency and stability of modified RNAs.
• Evaluation of fully modified RNAs lacking 2'-OH groups for CRISPR-Cas9 activity in human cells.

Main Results:

• Several heavily modified crRNA and tracrRNA variants demonstrated superior potency over unmodified counterparts.
• Fully chemically modified crRNAs and tracrRNAs, including those without 2'-OH groups, were functional in human cells.
• Increased RNA stability in vivo is associated with enhanced potency for modified guide RNAs.

Conclusions:

• Chemically modified guide RNAs significantly improve the performance of CRISPR-Cas9 systems.
• These modified RNAs offer enhanced stability and potency, crucial for effective in vivo and ex vivo gene editing.
• The developed modified RNA designs hold promise for advancing CRISPR-Cas9-based therapeutics, particularly with RNP and mRNA delivery methods.