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CRISPR-Cas9 gene therapy faces several key limitations that researchers are actively working to address. Here's a breakdown:

• Off-target effects: This refers to unintended DNA modifications at sites other than the intended target. The CRISPR-Cas9 system can sometimes cut DNA at locations with similar sequences to the target, potentially causing harmful mutations. Current rates vary from 0.1-5% depending on guide RNA design and delivery method.
• Delivery challenges: Getting CRISPR components into target cells efficiently remains difficult, particularly for in-vivo applications. Viral vectors (AAV) have size limitations, while lipid nanoparticles face issues with tissue specificity and immune responses.
• Immune responses: The Cas9 protein, derived from bacteria, can trigger immune reactions in patients. Pre-existing immunity to common Cas9 variants (from S. pyogenes and S. aureus) has been detected in 58-79% of humans.

By combining improved guide RNA design, novel delivery mechanisms, and next-generation editing tools like prime editing, researchers are making significant progress in overcoming these limitations. Prime editing (2023-24) has shown 90% reduction in off-target effects by using a nick-and-write mechanism rather than double-strand breaks.