Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

By Silvana Konermann, Mark D. Brigham, Alexandro E. Trevino, J. Keith Joung, Omar Abudayyeh, Clea Bárcena, P. Hsu, Naomi Habib, Jonathan Gootenberg, H. Nishimasu and 2 more. Nature, 2014

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Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/cd0bafc6298d34d66a43a217f187f2ac8ab5086c

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Citations in the corpus

RNA N6‐methyladenosine methyltransferase‐like 3 promotes liver cancer progression through YTHDF2‐dependent posttranscriptional silencing of SOCS2 (2018, methodology): “METTL3 activation in MHCC97L and Huh-7 cells was performed by using the CRISPR/dCas9-VP64/MS2-p65-HSF1 gene activation system.((33)) The shRNAs and singleguided RNAs used in this study are listed in Supporting Table S1.”
Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation (2016, background): “…while several strategies have been described for CRISPR-mediated activation (Chavez et al., 2015; Gilbert et al., 2013; Hilton et al., 2015; Konermann et al., 2015; Maeder et al., 2013; PerezPinera et al., 2013; Tanenbaum et al., 2014; Zalatan et al., 2015), a recent comparison of these…”
YY1 Is a Structural Regulator of Enhancer-Promoter Loops (2017, positive): “A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript # 61425) (Konermann et al., 2015)).”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “All of these studies show that synergistically recruiting multiple activators to the dCas9 target locus enhances the activation of the CRISPRa system (180, 184, 185).”
A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation1[OPEN] (2015, background): “SSNs can induce sequence-specific DNA double-strand breaks (DSBs), which are subsequently repaired by either nonhomologous end joining (NHEJ) or homologous recombination (Kanaar et al., 1998; Puchta, 2005).”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “…many areas, including identifying and understanding mechanisms of genetic diseases (Findlay et al., 2014; Gilbert et al., 2014; Zhou et al., 2014; Konermann et al., 2015), validating disease targets (Shalem et al., 2014; Wang et al., 2014), developing animal disease models (Wang et al., 2013;…”
Genome-wide Programmable Transcriptional Memory by CRISPR-based Epigenome Editing (2021, methodology): “When cells reached 60%–80% confluency the next day, cells were transfected with 500 ng of dCas9 plasmid (dCas9 or TETv1-4) and 300 ng of sgRNA-transactivator plasmid (sgRNA only, VP64, p65, Rta, VP64-p65, p65-Rta, or VPR) using TransIT-LT1 Transfection Reagent (Mirus) and Opti-MEMTM I Reduced SerumM”