Tracking genome engineering outcome at individual DNA breakpoints
Michael T. Certo,Byoung Y. Ryu,James Annis,Mikhail Garibov,Jordan Jarjour,Jordan Jarjour,David J. Rawlings,David J. Rawlings,Andrew M. Scharenberg,Andrew M. Scharenberg +9 more
TLDR
A genome engineering reporter system, designated 'traffic light', is presented that supports rapid flow-cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high-throughput screens for factors that bias the engineering outcome.Abstract:
Site-specific genome engineering technologies are increasingly important tools in the postgenomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks. Here we present a genome engineering reporter system, designated 'traffic light', that supports rapid flow-cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high-throughput screens for factors that bias the engineering outcome. We applied the traffic light system to evaluate the efficiency and outcome of nuclease-induced genome engineering in human cell lines and identified strategies to facilitate isolation of cells in which a desired engineering outcome has occurred.read more
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Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,Le Cong,F. Ann Ran,F. Ann Ran,David M. Cox,David M. Cox,Shuailiang Lin,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +15 more
TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.
Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,F. A. Ran,David Benjamin Turitz Cox,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +10 more
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
Journal ArticleDOI
CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.
Prashant Mali,John Aach,P. Benjamin Stranges,Kevin M. Esvelt,Mark Moosburner,Sriram Kosuri,Luhan Yang,George M. Church,George M. Church +8 more
TL;DR: This system is engineer to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA).
Journal ArticleDOI
A guide to genome engineering with programmable nucleases
Hyongbum Kim,Jin-Soo Kim +1 more
TL;DR: Known nuclease-specific features are essential for researchers to choose the most appropriate tool for a range of applications, including their composition, targetable sites, specificities and mutation signatures, among other characteristics.
Journal ArticleDOI
Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells
Van Trung Chu,Timm Weber,Benedikt Wefers,Wolfgang Wurst,Sandrine Sander,Klaus Rajewsky,Ralf Kühn +6 more
TL;DR: E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines and provide useful tools to improve the frequency of precise gene modifications in mammalian cells.
References
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