Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications

Didovyk, Andriy; Borek, Bartłomiej; Tsimring, Lev S.; Hasty, Jeff

doi:10.1016/j.copbio.2016.06.003

Cited by 72 publications

(42 citation statements)

References 72 publications

(119 reference statements)

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“…Among synthetic gene activators, dCas9-TADs potentially offer unparalleled simplicity and multiplexability compared to ZFP-TADs and TALE-TADs because sgRNAs can be easily modified to achieve new targeting specificities and dCas9 guided by multiple sgRNAs can simultaneously bind to several different target loci 10 . However, a dCas9 fusion with VP64, a frequently used TAD 11 , only weakly activates target gene using a single sgRNA in plant and mammalian cells 7–9,12–15 .…”

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confidence: 99%

A potent Cas9-derived gene activator for plant and mammalian cells

et al. 2017

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Overexpression of complementary DNA (cDNA) represents the most commonly used gain-of-function approach for interrogating gene functions and for manipulating biological traits. However, this approach is challenging and inefficient for multigene expression due to increased labor for cloning, limited vector capacity, requirement of multiple promoters and terminators, and variable transgene expression levels. Synthetic transcriptional activators provide a promising alternative strategy for gene activation by tethering an autonomous transcription activation domain (TAD) to an intended gene promoter at endogenous genomic locus through a programmable DNA-binding module. Among the known custom DNA-binding modules, the nuclease-dead Streptococcus pyogenes Cas9 (dCas9) protein, which recognizes a specific DNA target through base pairing between a synthetic guide RNA (sgRNA) and DNA, outperforms zinc finger proteins (ZFPs) and transcription activator-like effectors (TALEs), both of which target through protein-DNA interactions1. Recently, three potent dCas9-based transcriptional activation systems, namely VPR, SAM, and Suntag, have been developed for animal cells2–6. However, an efficient dCas9-based transcriptional activation platform is still lacking for plant cells7–9. Here, we developed a new potent dCas9-TAD named dCas9-TV through plant cell-based screens, which confers far stronger transcriptional activation of a single or multiple target genes than the routinely used dCas9-VP64 activator in both plant and mammalian cells.

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confidence: 99%

A potent Cas9-derived gene activator for plant and mammalian cells

et al. 2017

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“…Since then, efforts have been made to expand the set of DNA-binding proteins for the design of more complex circuits, mainly focusing on repressor molecules7891011, although more recently several activators were developed1213. Nonetheless, there are fewer activators and indeed sets of dual activator–repressor or repressor–repressor switches have not been reported thus far, even though these would be very useful for engineering devices.…”

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confidence: 99%

Engineering orthogonal dual transcription factors for multi-input synthetic promoters

2016

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Synthetic biology has seen an explosive growth in the capability of engineering artificial gene circuits from transcription factors (TFs), particularly in bacteria. However, most artificial networks still employ the same core set of TFs (for example LacI, TetR and cI). The TFs mostly function via repression and it is difficult to integrate multiple inputs in promoter logic. Here we present to our knowledge the first set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants and multi-input promoter architectures. Our toolkit contains 12 TFs, flexibly operating as activators, repressors, dual activator–repressors or dual repressor–repressors, on up to 270 synthetic promoters. To engineer non cross-reacting cI variants, we design a new M13 phagemid-based system for the directed evolution of biomolecules. Because cI is used in so many synthetic biology projects, the new set of variants will easily slot into the existing projects of other groups, greatly expanding current engineering capacities.

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“…Because our method is applicable to both the catalytically active and dead versions of the nuclease, it should also lead to improvements in a vast range of CRISPR applications, including in vivo gene editing, programmable repression, and nucleic acid detection. Our multiplexed approach is particularly applicable to the advancement of dCas-based gene circuit elements, which can be been used to create complex circuits that behave orthogonally, operating independently without crosstalk [47][48][49][50][51]. Furthermore, our approach can expedite the rational design of enhanced CRISPR nucleases and facilitate the development of CRISPR-Cas variants with greater specificity, improved proofreading capabilities, or increased activities [52][53][54][55][56][57].…”

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confidence: 99%

Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

Specht

Lambert

2019

Preprint

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The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has lead to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable non-specific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of Francisella novicida Cas12a (FnCas12a) as it searches for its DNA target. Our results reveal concealed thermodynamic factors affecting FnCas12a DNA binding which should guide the design and optimization of crRNA that limit off-target effects, including the crucial role of an extended PAM sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.

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Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications

Cited by 72 publications

References 72 publications

A potent Cas9-derived gene activator for plant and mammalian cells

A potent Cas9-derived gene activator for plant and mammalian cells

Engineering orthogonal dual transcription factors for multi-input synthetic promoters

Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

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