Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9
A peer-reviewed article of this Preprint also exists.
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Abstract
CRISPR/Cas9 technology enables the rapid and efficient generation of total loss-of- function mutations in a targeted gene in mammalian cells. A single cell that harbors those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of various biological reagents. However, the successful derivation of knockout clones may be technically challenging and can be complicated by multiple factors, including incomplete target ablation and inter-clonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation and identification of true knockout clones and that are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells.
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2019. Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9. PeerJ Preprints 7:e27511v1 https://doi.org/10.7287/peerj.preprints.27511v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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This is a preprint submission to PeerJ Preprints.
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Competing Interests
The authors declare no competing interests.
Author Contributions
Christopher J Giuliano conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Ann Lin conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Jason Sheltzer conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Funding
Research in the Sheltzer Lab is supported by an NIH Early Independence Award (1DP5OD021385), a Breast Cancer Alliance Young Investigator Award, a Damon Runyon- Rachleff Innovation Award, a Gates Foundation Innovative Technology Solutions grant, and a CSHL-Northwell Translational Cancer Research Grant. This work was performed with assistance from the CSHL Flow Cytometry Shared Resource, which is supported by the CSHL Cancer Center Support Grant 5P30CA045508. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
