Miniaturized scalable arrayed CRISPR screening in primary cells enables discovery at the single donor resolution

Area of Science:

• Biotechnology
• Molecular Biology
• Genomics

Background:

• Efficient gene editing in primary human cells is crucial for therapeutics and functional genomics.
• Conventional electroporation methods often demand high cell numbers and lack scalability for parallel experiments.

Purpose of the Study:

• To develop and validate a next-generation digital microfluidics (DMF) electroporation platform for high-throughput, low-input genome engineering.
• To enable efficient delivery of CRISPR-Cas9 ribonucleoprotein complexes (RNPs) and mRNA into primary human cells.
• To demonstrate the platform's utility in functional genomics screens using rare or precious cell types.

Main Methods:

• Utilized a digital microfluidics (DMF) platform with a planar electrode array for manipulating discrete droplets.
• Integrated the DMF system with laboratory automation for parallelized experiments.
• Delivered CRISPR-Cas9 RNPs and mRNA cargo into primary human cells at low input levels (as few as 3,000 cells/condition).

Main Results:

• Demonstrated high-efficiency transfection and gene editing across diverse primary human cell types.
• Achieved high rates of gene knockout via non-homologous end joining (NHEJ) and precise knock-in via homology-directed repair (HDR).
• Successfully applied the platform to an arrayed CRISPR-Cas9 screen in CD4+ T cells, identifying novel regulators of T cell exhaustion.

Conclusions:

• The DMF electroporation platform offers a powerful, miniaturized solution for genome engineering in rare and precious primary human cells.
• The system provides a scalable framework for high-content genetic screening, advancing therapeutic development and functional genomics research.