title: Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9.
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.
authors.affiliation: Department of Biomedical Engineering, University of North Carolina, Chapel Hill NC and North Carolina State University, Raleigh, NC, United States.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Attayek
authors.affiliation: Department of Medicine, University of North Carolina, Chapel Hill, NC, United States.
authors.forename: Jennifer P
authors.initials: JP
authors.lastname: Waugh
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, United States.
authors.forename: Sally A
authors.initials: SA
authors.lastname: Hunsucker
authors.affiliation: Department of Medicine, University of North Carolina, Chapel Hill, NC, United States.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Grayeski
authors.affiliation: Department of Medicine, University of North Carolina, Chapel Hill, NC, United States; Department of Chemistry, University of North Carolina, Chapel Hill, NC, United States.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.affiliation: Department of Medicine, University of North Carolina, Chapel Hill, NC, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, United States.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Armistead
authors.affiliation: Department of Biomedical Engineering, University of North Carolina, Chapel Hill NC and North Carolina State University, Raleigh, NC, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, United States; Department of Chemistry, University of North Carolina, Chapel Hill, NC, United States. Electronic address: nlallbri@unc.edu.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D064113:CRISPR-Cas Systems; D004867:Equipment Design; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007091:Image Processing, Computer-Assisted; D020014:K562 Cells; D007938:Leukemia / Q000235:genetics; D009154:Mutation; D000072668:Splicing Factor U2AF / Q000235:genetics*; D046888:Tissue Array Analysis / Q000295:instrumentation* / Q000379:methods; D014162:Transfection
chemical_list: D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: CRISPR-Cas9; Cell array; Cell sorting; Cytometry; Microraft