Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9

- Microraft array platform enables monitoring of temporal cell characteristics.
- Achieved automated identification and retrieval of living cells from a microarray (microraft) platform.
- Individual living cells were identified and retrieved with high sensitivity and efficiency.
- Automated imaging of microraft array reveals GFP temporal expression.
- Automated microraft screening efficiently identified CRISPR Cas9 gene-engineered cell lines.

Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation.