Single step, high efficiency CRISPR-Cas9 genome editing in primary human disease-derived fibroblasts
Robert B Good,
Carmel B Nanthakumar,
Andy D Blanchard,
Posted 11 Oct 2018
bioRxiv DOI: 10.1101/440099 (published DOI: 10.1089/crispr.2018.0047)
Posted 11 Oct 2018
Genome editing is a tool that has many applications including the validation of potential drug targets. However, performing genome editing in low passage, primary human cells with the greatest physiological relevance, is notoriously difficult. High editing efficiency is desired because it enables gene knock outs (KO) to be generated in bulk cellular populations and circumvents the problem of having to generate clonal cell isolates. Here, we describe a single step workflow enabling >90% KO generation in primary human lung fibroblasts via CRISPR ribonucleoprotein delivery, in the absence of antibiotic selection or clonal expansion. As proof of concept, we performed a disease relevant phenotypic assay measuring collagen deposition in response to TGFβ and demonstrated SMAD3 but not SMAD2 dependent deposition of type I collagen following knockout of each using our single step methodology. The optimization of this workflow can readily be transferred to other primary cell types.
- Downloaded 1,624 times
- Download rankings, all-time:
- Site-wide: 6,180 out of 103,802
- In molecular biology: 215 out of 3,584
- Year to date:
- Site-wide: 10,386 out of 103,802
- Since beginning of last month:
- Site-wide: 8,857 out of 103,802
Downloads over time
Distribution of downloads per paper, site-wide
- 18 Dec 2019: We're pleased to announce PanLingua, a new tool that enables you to search for machine-translated bioRxiv preprints using more than 100 different languages.
- 21 May 2019: PLOS Biology has published a community page about Rxivist.org and its design.
- 10 May 2019: The paper analyzing the Rxivist dataset has been published at eLife.
- 1 Mar 2019: We now have summary statistics about bioRxiv downloads and submissions.
- 8 Feb 2019: Data from Altmetric is now available on the Rxivist details page for every preprint. Look for the "donut" under the download metrics.
- 30 Jan 2019: preLights has featured the Rxivist preprint and written about our findings.
- 22 Jan 2019: Nature just published an article about Rxivist and our data.
- 13 Jan 2019: The Rxivist preprint is live!