Deep Learning‐Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard‐to‐Transfect Cells

Abstract

Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human‐induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP‐E) system, a nanopipette‐based single‐cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell‐nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP‐E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.

A Review of Single‐Cell Pose Adjustment and Puncture

“…Transformation frequency in terms of plants grown to maturity and showing the presence of the introduced genes was 1.4%. In addition, Ukherjee et al [ 195 ] proposed a single‐cell electroporation method based on nanopipettes, which showed superior cell viability and efficiency compared with traditional methods. The automated system uses deep convolutional networks to identify cell locations.…”

The new era of high throughput nanoelectrochemistry

“…[141][142][143][144][145] Despite these studies being linked to high-throughput downstream biological analysis, the number of collectable samples is still limited by the lack of automation. Mukherjee et al 146 developed a fully automated nanofountain probe electroporation system based on angular approach SICM. 147 A fully convolutional network was used to spatially localize cells in the optical field of view and to tag the position of nuclei.…”

“…Automation partially overcomes some of the key problems with manual probe-based approaches, allowing the manipulation of tens of cells with the same nanopipette. 146 Hu et al used an Al2O3-coated nanopipette as an SICM probe to detect intracellular reactive oxygen species (ROS). Their system is based on a computer-vision algorithm for automatic detection of cell nuclei and lateral nanopipette positioning.…”

Multiplexed high-throughput localized electroporation workflow with deep learning–based analysis for cell engineering

“…In addition, some of these systems may suffer from biofouling or clogging issues. On the other hand, micro-and nanoprobe-based methods use micro/nanopipettes (23)(24)(25)(26) or hollow atomic force microscopy tips (such as the nanofountain probe and fluidic force microscopy) (27,28) for precisely controlled delivery into cells with subcellular resolution. The major drawbacks of these systems are their serial approach that limits throughput.…”