cloning-free crispr

Clicks: 61
ID: 223407
2015
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Ranked #100 of 101 articles by views in nature reviews gastroenterology & hepatology

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Abstract
We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.
Reference Key
arbab2015stemcloning-free Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors ;Mandana Arbab;Sharanya Srinivasan;Tatsunori Hashimoto;Niels Geijsen;Richard I. Sherwood
Journal nature reviews gastroenterology & hepatology
Year 2015
DOI
10.1016/j.stemcr.2015.09.022
URL
http://www.sciencedirect.com/science/article/pii/S2213671115002842
Keywords
medicine (general)biology (general)

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