Hairpin Internal Nuclear Localization Signals in CRISPR-Cas9 Enhance Editing in Primary Human Lymphocytes

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Cellular Engineering

Background:

• Nuclear localization signal (NLS) sequences are crucial for directing CRISPR enzymes into the cell nucleus for genome editing.
• Terminal NLS fusions enhance CRISPR enzyme performance but can reduce protein yield during recombinant expression.
• Optimizing CRISPR enzyme delivery and efficiency remains a key challenge in gene editing applications.

Purpose of the Study:

• To develop a novel strategy for enhancing CRISPR-Cas9 genome editing efficiency and protein production.
• To investigate the efficacy of hairpin internal NLS (hiNLS) sequences integrated within the CRISPR-Cas9 backbone.
• To compare the performance of hiNLS Cas9 variants against terminally fused NLS constructs in human T cells.

Main Methods:

• Engineered CRISPR-Cas9 variants with hairpin internal NLS (hiNLS) sequences were designed and constructed.
• hiNLS Cas9 variants were evaluated for gene editing efficiency in human primary T cells.
• Delivery of ribonucleoprotein complexes was achieved via electroporation and co-incubation with amphiphilic peptides.

Main Results:

• hiNLS Cas9 variants demonstrated improved gene editing efficiency in human T cells compared to terminal NLS fusions.
• Many hiNLS Cas9 constructs exhibited high purity and yield, even with multiple NLS insertions (up to nine).
• The hiNLS strategy successfully facilitated nuclear localization and efficient DNA editing.

Conclusions:

• Hairpin internal NLS (hiNLS) sequences represent a significant advancement in CRISPR effector design.
• This approach overcomes limitations of terminal NLS fusions, improving both editing efficiency and protein yield.
• hiNLS Cas9 constructs hold promise for enhanced genome editing outcomes in research and therapeutic applications.