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Commun Biol. 2018 May 3;1:41. doi: 10.1038/s42003-018-0034-6. eCollection 2018.

Light-activated cell identification and sorting (LACIS) for selection of edited clones on a nanofluidic device.

Author information

1
Berkeley Lights, Inc, 5858 Horton Street #320, Emeryville, CA, 94608, USA.
2
UCSF Department of Microbiology and Immunology, University of California, San Francisco, CA, 94143, USA.
3
UCSF Department of Microbiology and Immunology, University of California, San Francisco, CA, 94143, USA. Alexander.Marson@UCSF.edu.
4
Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA. Alexander.Marson@UCSF.edu.
5
Diabetes Center, University of California, San Francisco, CA, 94143, USA. Alexander.Marson@UCSF.edu.
6
Innovative Genomics Institute, University of California, Berkeley, CA, 94720, USA. Alexander.Marson@UCSF.edu.
7
Department of Medicine, University of California, San Francisco, CA, 94143, USA. Alexander.Marson@UCSF.edu.
8
UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, 94158, USA. Alexander.Marson@UCSF.edu.
9
Berkeley Lights, Inc, 5858 Horton Street #320, Emeryville, CA, 94608, USA. Gregory.lavieu@curie.fr.
10
Institut Curie, INSERM U932-Immunity and Cancer, Paris, 75005, France. Gregory.lavieu@curie.fr.

Abstract

Despite improvements in the CRISPR molecular toolbox, identifying and purifying properly edited clones remains slow, laborious, and low-yield. Here, we establish a method to enable clonal isolation, selection, and expansion of properly edited cells, using OptoElectroPositioning technology for single-cell manipulation on a nanofluidic device. Briefly, after electroporation of primary T cells with CXCR4-targeting Cas9 ribonucleoproteins, single T cells are isolated on a chip and expanded into colonies. Phenotypic consequences of editing are rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, individual colonies are identified based on their specific genotype. Each colony is split and sequentially exported for on-target sequencing and further off-chip clonal expansion of the validated clones. Using this method, single-clone editing efficiencies, including the rate of mono- and bi-allelic indels or precise nucleotide replacements, can be assessed within 10 days from Cas9 ribonucleoprotein introduction in cells.

Conflict of interest statement

The authors declare the following competing interests: An.M., H.M.B., M.S., A.S., K.C., and G.L. are or were fulltime employees of Berkeley Lights, Inc, a private company. Al.M. is a founder of Spotlight Therapeutics and serves as an advisor to Juno Therapeutics and PACT Pharma and the Marson lab has received sponsored research support from Juno Therapeutics, Epinomics, and Sanofi. The remaining authors declare no competing interests.

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