Dual-reporter surrogate systems for efficient enrichment of genetically modified cells

Cellular and Molecular Life Sciences : CMLS
Chonghua RenZhiying Zhang

Abstract

Isolation of genetically modified cells generated by designed nucleases are challenging, since they are often phenotypically indistinguishable from their parental cells. To efficiently enrich genetically modified cells, we developed two dual-reporter surrogate systems, namely NHEJ-RPG and SSA-RPG based on NHEJ and SSA repair mechanisms, respectively. Repair and enrichment efficiencies of these two systems were compared using different nucleases. In both CRISPR-Cas9- and ZFNs-induced DSB repair studies, we found that the efficiency and sensitivity of the SSA-RPG reporter with direct repeat length more than 200 bp were much higher than the NHEJ-RPG reporter. By utilizing the SSA-RPG reporter, we achieved the enrichment for indels in several endogenous loci with 6.3- to 34.8-fold of non-selected cells. Thus, the highly sensitive SSA-RPG reporter can be used for activity validation of designed nucleases and efficient enrichment of genetically modified cells. Besides, our systems offer alternative enrichment choices either by puromycin selection or FACS.

References

Dec 24, 2003·Analytical Biochemistry·Andreas Seyfang, Jean Huaqian Jin
Aug 3, 2005·BioTechniques·Christophe PerezPhilippe Duchateau
Nov 22, 2005·Genetics·Christine R PrestonWilliam R Engels
Oct 25, 2006·Proceedings of the National Academy of Sciences of the United States of America·Jason MortonDana Carroll
Jun 12, 2008·Molecular Therapy : the Journal of the American Society of Gene Therapy·Toni Cathomen, J Keith Joung
Jan 9, 2010·Science·Philippe Horvath, Rodolphe Barrangou
Aug 4, 2010·Methods in Molecular Biology·Dmitry Y GuschinEdward J Rebar
May 17, 2011·BMC Bioinformatics·Thomas J CradickAnton P McCaffrey
Oct 11, 2011·Nature Methods·Hyojin KimJin-Soo Kim
Feb 18, 2012·Nature·Blake WiedenheftJennifer A Doudna
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Sep 6, 2012·Proceedings of the National Academy of Sciences of the United States of America·Giedrius GasiunasVirginijus Siksnys
Oct 2, 2012·Journal of Genetics and Genomics = Yi Chuan Xue Bao·Jingang LiuJun Chen
Dec 15, 2012·Science·Elizabeth Pennisi
Jan 5, 2013·Science·Le CongFeng Zhang
Jan 5, 2013·Science·Prashant MaliGeorge M Church
Jan 31, 2013·Nature Biotechnology·Woong Y HwangJ Keith Joung
Feb 26, 2013·International Journal of Radiation Oncology, Biology, Physics·Chen Wang, Susan P Lees-Miller
May 21, 2013·DNA Repair·Teruaki Iyama, David M Wilson
May 25, 2013·Annual Review of Genomics and Human Genetics·David J Segal, Joshua F Meckler
May 28, 2013·Genetics·Scott J GratzKate M O'Connor-Giles
Jul 3, 2013·Nature Methods·Ari E FriedlandJohn A Calarco
Feb 27, 2014·Nature Communications·Suresh RamakrishnaHyongbum Kim
May 2, 2014·Methods : a Companion to Methods in Enzymology·Young-Hoon KimJin-Soo Kim
May 3, 2014·Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology·Alexander KnollHolger Puchta
Jul 21, 2014·Cellular and Molecular Life Sciences : CMLS·Kun XuZhiying Zhang
Jul 1, 2008·Molecular Therapy : the Journal of the American Society of Gene Therapy·Toni Cathomen, J Keith Joung

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Citations

Jan 13, 2016·Cellular and Molecular Life Sciences : CMLS·Yan ZhouYonglun Luo
Aug 11, 2018·Reviews in Medical Virology·Garry A Luke, Martin D Ryan
Aug 8, 2018·The FEBS Journal·Xinyi LiZhiying Zhang
Sep 14, 2018·Applied Microbiology and Biotechnology·Zhixin LuoXin Wang
Apr 20, 2017·Cellular and Molecular Life Sciences : CMLS·Francesca NiccheriSilvestro G Conticello
Feb 13, 2016·G3 : Genes - Genomes - Genetics·Yichun BaiZhiying Zhang
Mar 7, 2021·International Journal of Molecular Sciences·Janusz Blasiak

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