Highly efficient CRISPR-mediated genome editing through microfluidic droplet cell mechanoporation

Area of Science:

• Biotechnology
• Molecular Biology
• Bioengineering

Background:

• CRISPR-Cas9 technology has revolutionized genome editing but faces limitations due to inefficient and unsafe delivery methods.
• Current delivery techniques often struggle with efficiency and precision, hindering therapeutic applications.

Purpose of the Study:

• To develop a novel, highly efficient, and safe microfluidic system for CRISPR gene delivery.
• To overcome the limitations of existing CRISPR delivery methods for therapeutic and research applications.

Main Methods:

• Introduction of the droplet cell pincher (DCP), a microfluidic system combining droplet microfluidics and cell mechanoporation.
• Encapsulation and controlled passage of cells and CRISPR components through a microscale constriction to facilitate membrane discontinuity and nuclear entry.
• Demonstration of macromolecule delivery, including mRNA and plasmid DNA, and subsequent CRISPR-Cas9 genome engineering.

Main Results:

• Achieved high delivery efficiency for mRNA (~98%) and plasmid DNA (~91%) using the DCP platform.
• Demonstrated superior performance over electroporation in CRISPR-Cas9 mediated single knockouts (~6.5-fold), double knockouts (~3.8-fold), and knock-ins (~3.8-fold).

Conclusions:

• The droplet cell pincher (DCP) system represents a significant advancement in CRISPR delivery technology.
• This platform shows great potential as a next-generation tool for CRISPR engineering in clinical and biological research.