[Establishment and Application of Efficient Gene Editing Method for Classical HLA-I Molecules].

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Immunology

Context:

• Human Leukocyte Antigen class I (HLA-I) molecules play a critical role in immune recognition and transplantation compatibility.
• Current gene editing methods face challenges in efficiently targeting and modifying hematopoietic stem cells.
• Developing universal HLA-I negative stem cells is crucial for reducing graft-versus-host disease and improving transplant outcomes.

Purpose:

• To establish an efficient gene editing method for the human leukocyte antigen class I (HLA-I) gene.
• To prepare HLA-I universal hematopoietic stem cells through gene editing.
• To optimize CRISPR-Cas9 RNP complex parameters for efficient gene editing in stem cells.

Summary:

• Designed and synthesized easyedit small guide RNA (sgRNA) targeting the β2 microglobulin gene.
• Formed Ribonucleoprotein (RNP) complexes with NLS-Cas9-NLS nuclease and sgRNA at various molar ratios.
• Successfully achieved HLA-I negative expression in HEK-293 cells and CD34+ hematopoietic stem cells via nucleotransfection, with optimized ratios yielding high silencing rates (up to 91.56%) and cleavage efficiencies (up to 64%).

Impact:

• Provides an efficient gene editing strategy for silencing classical HLA-I molecules.
• Demonstrates the suitability of this method for challenging stem cell transfection systems.
• Enables the generation of universal HLA-I negative stem cells for potential therapeutic applications in transplantation.