Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR systems are powerful tools for targeted gene expression regulation and creating genetically modified organisms.
• Traditional CRISPR/Cas9 plasmid delivery methods have drawbacks due to the presence of exogenous DNA within cells.
• Ribonucleoprotein (RNP) complexes offer a promising alternative for CRISPR delivery, enabling better control and fewer off-target mutations.

Purpose of the Study:

• To review strategies for improving synthetic guide RNA (sgRNA) function in CRISPR gene editing.
• To highlight the benefits of nucleotide modifications in synthetic sgRNAs.
• To discuss methods for enhancing sgRNA stability, efficacy, and reducing cellular toxicity.

Main Methods:

• Review of current literature on CRISPR/Cas9 delivery systems.
• Analysis of artificial RNA synthesis strategies for sgRNA modification.
• Examination of nucleotide modifications impacting RNA stability, binding affinity, and immunogenicity.

Main Results:

• Synthetic sgRNAs can be engineered with backbone alterations, native motifs, and labels.
• RNA modifications can increase resistance to hydrolysis and improve RNA-protein/RNA-DNA complex stability.
• Modified sgRNAs demonstrate reduced immunogenic and cytotoxic effects compared to unmodified versions.

Conclusions:

• Nucleotide modification of synthetic sgRNAs is a key strategy to enhance CRISPR gene editing efficiency and safety.
• The RNP delivery method combined with modified sgRNAs offers a superior alternative to plasmid-based CRISPR systems.
• Further research into synthetic sgRNA modifications will advance the application of genome editing technologies.