High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells
| Autor: | Meng Lou, Benjamin Steyer, Lucille Kohlenberg, Madelyn Goedland, Arezoo Movaghar, Jared Carlson-Stevermer, Krishanu Saha, Ryan Prestil |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Pluripotent Stem Cells Time Factors Cellular differentiation Human Embryonic Stem Cells Molecular Sequence Data Computational biology Biology Stem cell marker Biochemistry Article Cell Line 03 medical and health sciences Genome editing Genetics CRISPR Humans Induced pluripotent stem cell lcsh:QH301-705.5 Cell Proliferation lcsh:R5-920 Base Sequence Genome Human Gene targeting Gene Expression Regulation Developmental High-Throughput Nucleotide Sequencing Reproducibility of Results Cell Differentiation Cell Biology Embryonic stem cell 3. Good health 030104 developmental biology lcsh:Biology (General) High-content screening Gene Targeting CRISPR-Cas Systems lcsh:Medicine (General) Developmental Biology |
| Zdroj: | Stem Cell Reports, Vol 6, Iss 1, Pp 109-120 (2016) Stem Cell Reports |
| ISSN: | 2213-6711 |
| Popis: | Summary CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing. Graphical Abstract Highlights • High-content analysis of arrayed hESC colonies increased gene-editing efficiency • Rapid one-pot transcription of sgRNAs can be multiplexed to edit hESCs • hESCs gene edited on ArrayEdit exhibited proper phenotypes • ArrayEdit provides a new window into the process of gene editing human cells Saha and colleagues show that a new culture and imaging platform, called ArrayEdit, allows both high-throughput and high-resolution subcellular imaging of thousands of hESC colonies in parallel. ArrayEdit generated CRISPR-Cas9-edited, living hESC lines at 82% efficiency within days. Biallelic edited cell lines at the LAMA5 locus did not contain any detectable off-target mutations and exhibited expected self-renewal defects upon subsequent characterization. |
| Databáze: | OpenAIRE |
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