CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol

1] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [2].
Nature Biotechnology (Impact Factor: 41.51). 08/2013; 31(9). DOI: 10.1038/nbt.2675
Source: PubMed


Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system 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). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

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Available from: Prashant Mali, Aug 13, 2014
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    • "Our results show that increasing the number of VP16 transactivator repeats fused to dCas9 leads to improved activation of the endogenous OCT4. Using dCas9VP192 and five gRNAs, we obtained up to 70-fold OCT4 upregulation in HEK293 cells, in similar range of efficiency with previous studies (Cheng et al., 2013; Hu et al., 2014; Mali et al., 2013b). The fact that dCas9 activators can replace transgenic OCT4 as a reprogramming factor in human iPSC derivation speaks for the functional relevance of the gene activation and the potential for utilizing this approach for human cell type conversions. "
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    ABSTRACT: CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.
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    • "However, this may become a limiting factor if an alternative strategy using modified Cas9 " nickase " is utilized. While the main advantage of the Cas9 nickase is to greatly reduce the offtarget activity of the Cas9 endonuclease, it utilizes paired gRNAs to create targeted double strand breaks in the genome (Mali et al., 2013a; Ran et al., 2013). Large deletions described here would then require four gRNAs (i.e. "
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    ABSTRACT: Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model. Copyright © 2015 Elsevier Inc. All rights reserved.
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    • "In addition, CRISPR-mediated genome editing could offer significant clinical therapeutic potential by identifying particular genes involved in mediating dysregulated stress responsiveness in rodents (Mali et al, 2013). CRISPR is an innovative research tool that produces selective gene editing for effective manipulation of gene expression in the endogenous environment and overcomes some of the limitations of previous genome modifying methodologies. "
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    ABSTRACT: The normal function of the hypothalamic-pituitary-adrenal (HPA) axis, and resultant glucocorticoid (GC) secretion, is essential for human health. Disruption of GC regulation is associated with both pathologic, psychological and physiological disease states such as depression, post-traumatic stress disorder, hypertension, diabetes, and osteopenia, amongst others. As such, understanding the mechanisms by which HPA output is tightly regulated in its responses to environmental stressors and circadian cues has been an active area of investigation for decades. Over the last 20 years, however, advances in gene targeting and genome modification in rodent models has allowed the detailed dissection of roles for key molecular mediators and brain regions responsible for this control in vivo to emerge. Here, we summarize work done to elucidate the function of critical neuropeptide systems, GC-signaling targets, and inflammation-associated pathways in HPA axis regulation and behavior, and highlight areas for future investigation.Neuropsychopharmacology accepted article preview online, 20 July 2015. doi:10.1038/npp.2015.215.
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