Person: Linder, Sam
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Linder
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Sam
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Linder, Sam
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Publication In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites(Oxford University Press, 2013) Sander, Jeffry D.; Ramirez, Cherie Lynn; Linder, Sam; Pattanayak, Vikram; Shoresh, Noam; Ku, Manching; Foden, Jennifer A.; Reyon, Deepak; Bernstein, Bradley; Liu, David; Joung, J. KeithGene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.Publication Robust, synergistic regulation of human gene expression using TALE activators(2013) Maeder, Morgan L.; Linder, Sam; Reyon, Deepak; Angstman, James; Fu, Yanfang; Sander, Jeffry D.; Joung, J. KeithArtificial transcription activator-like (TAL) effector-based activators (TALE activators) have broad utility but previous studies suggest that these monomeric proteins often possess low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy and guide design of novel monomeric TAL effector-based fusion proteins.Publication CRISPR RNA-guided activation of endogenous human genes(2013) Maeder, Morgan L; Linder, Sam; Cascio, Vincent M; Fu, Yanfang; Ho, Quan H; Joung, J KeithCatalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells.Publication Targeted DNA Demethylation and Endogenous Gene Activation Using Programmable TALE-TET1 Fusions(2013) Maeder, Morgan L; Angstman, James; Richardson, Marcy E; Linder, Sam; Cascio, Vincent M; Tsai, Shengdar Q.; Ho, Quan H; Sander, Jeffry D; Reyon, Deepak; Bernstein, Bradley; Costello, Joseph F; Wilkinson, Miles F; Joung, J Keith