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

Peter J. Attayek, Jennifer P. Waugh, Sally A. Hunsucker, Philip J. Grayeski, Christopher E. Sims, Paul M. Armistead, Nancy L. Allbritton

Research output: Research - peer-reviewArticle

Abstract

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.

LanguageEnglish (US)
Pages175-182
Number of pages8
JournalBiosensors and Bioelectronics
Volume91
DOIs
StatePublished - May 15 2017

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Genes
Hardware
Software
Screening
Throughput
Imaging techniques
Mutation
Time-Lapse Imaging
Technology
Gene Editing

Keywords

  • Cell array
  • Cell sorting
  • CRISPR-Cas9
  • Cytometry
  • Microraft

ASJC Scopus subject areas

  • Biotechnology
  • Biophysics
  • Biomedical Engineering
  • Electrochemistry

Cite this

Attayek, P. J., Waugh, J. P., Hunsucker, S. A., Grayeski, P. J., Sims, C. E., Armistead, P. M., & Allbritton, N. L. (2017). Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9. Biosensors and Bioelectronics, 91, 175-182. DOI: 10.1016/j.bios.2016.12.019

Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9. / Attayek, Peter J.; Waugh, Jennifer P.; Hunsucker, Sally A.; Grayeski, Philip J.; Sims, Christopher E.; Armistead, Paul M.; Allbritton, Nancy L.

In: Biosensors and Bioelectronics, Vol. 91, 15.05.2017, p. 175-182.

Research output: Research - peer-reviewArticle

Attayek, PJ, Waugh, JP, Hunsucker, SA, Grayeski, PJ, Sims, CE, Armistead, PM & Allbritton, NL 2017, 'Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9' Biosensors and Bioelectronics, vol 91, pp. 175-182. DOI: 10.1016/j.bios.2016.12.019
Attayek PJ, Waugh JP, Hunsucker SA, Grayeski PJ, Sims CE, Armistead PM et al. Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9. Biosensors and Bioelectronics. 2017 May 15;91:175-182. Available from, DOI: 10.1016/j.bios.2016.12.019
Attayek, Peter J. ; Waugh, Jennifer P. ; Hunsucker, Sally A. ; Grayeski, Philip J. ; Sims, Christopher E. ; Armistead, Paul M. ; Allbritton, Nancy L./ Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9. In: Biosensors and Bioelectronics. 2017 ; Vol. 91. pp. 175-182
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