Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a

Area of Science:

• Biotechnology
• Molecular Biology
• Immunotherapy

Background:

• Chimeric antigen receptor (CAR)-T cell therapy is a promising cancer treatment.
• Efficient and precise genetic engineering of T cells is crucial for CAR-T cell development.
• Existing methods for CAR gene integration can be inefficient or lack specificity.

Purpose of the Study:

• To develop a novel system for generating human CAR-T cells.
• To achieve site-specific and efficient integration of CAR genes into the T cell genome.
• To utilize a conjugated Cas12a (cCas12a) system for precise genome editing.

Main Methods:

• Site-specific modification of Cas12a enzyme.
• Preparation of Cas12a-CRISPR RNA (crRNA) complex using bio-orthogonal chemistry.
• CAR-T cell generation via electroporation and adeno-associated virus (AAV) infection.

Main Results:

• Demonstrated robust editing efficiency in human cells.
• Achieved precise and highly efficient integration of CAR genes into the T cell genome.
• Successfully generated human CAR-T cells using the cCas12a system.

Conclusions:

• The developed cCas12a system provides a powerful tool for CAR-T cell engineering.
• This protocol enables precise, efficient, and targeted CAR gene integration.
• The system holds significant potential for advancing CAR-T cell therapy development.