CRISPR-mediated transcriptional activation with synthetic guide RNA

Strezoska, Žaklina; Dickerson, Sarah; Maksimova, Elena; Chou, Eldon T.; Gross, Maren M.; Hemphill, Kevin; Hardcastle, Travis; Perkett, Matthew R.; Stombaugh, Jesse; Miller, Galen W.; Anderson, Emily M.; Vermeulen, Annaleen; Smith, Anja van Brabant

doi:10.1016/j.jbiotec.2020.05.005

Cited by 16 publications

(11 citation statements)

References 43 publications

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“…1D ). When delivering just one of the two CXCR4 -targeting sgRNAs, we observed lower levels of activation and shorter activation duration, confirming prior studies showing the possibility of tuning these parameters with sgRNA selection ( Strezoska et al 2020 ).…”

Section: Resultssupporting

confidence: 87%

“…Here, single-cell RNA-seq from almost 204,000 cells expressing a unique sgRNA showed that only 49.6% of sgRNAs activated the cognate gene. The proportion of active sgRNAs is higher in our study, which might be owing to RNA delivery being more potent than lentiviral delivery, as has been shown previously ( Strezoska et al 2020 ). However, we were not able to induce ITGAX gene expression despite testing 10 different sgRNAs scattered within a 495-bp window from −633 bp to −138 bp relative to the TSS.…”

Section: Discussionsupporting

confidence: 78%

“…Compared with cDNA delivery approaches, RNA-based CRISPRa/i benefits from being cloning-free, and it induces or represses expression of all isoforms transcribed from a promoter. It is highly scalable and easily implemented for arrayed CRISPRa/i screens as has previously been described ( Strezoska et al 2020 ). Additionally, transcriptional engineering may find use in cellular therapeutics and regenerative medicine to direct cell differentiation to a specific lineage or to enhance specific cell functions.…”

Section: Discussionmentioning

confidence: 99%

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Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi

et al. 2021

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Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance due to lack of effective and non-toxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation, and show transient, programmable gene regulation in primary cells, including human CD34+ hematopoietic stem and progenitor cells (HSPCs) and human CD3+ T cells. We demonstrate multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.

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Section: Resultssupporting

confidence: 87%

Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

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Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi

et al. 2021

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“…Synthetic guide RNAs can be generated quickly and have proven effective for Cas9-mediated gene disruption purposes ranging from the creation of in vitro and in vivo models to arrayed genetic screens 26 . While synthetic gRNAs have previously been applied in the context of CRISPRa 27 thus far they have not been widely adopted possibly due to their low efficiency with sub-optimal CRISPRa systems. The production of synthetic, high-efficiency, SAM-compatible guides has presented technical challenges.…”

Section: Resultsmentioning

confidence: 99%

A versatile, high-efficiency platform for CRISPR-based gene activation

Heidersbach¹,

Dorighi²,

Gomez

et al. 2022

Preprint

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CRISPR-mediated transcriptional activation (CRISPRa) is a powerful technology for inducing gene expression from endogenous loci with exciting applications in high throughput gain-of-function genomic screens and the engineering of cell-based models. However, current strategies for generating potent, stable, CRISPRa-competent cell-lines present limitations for the broad utility of this approach. Here, we provide a high-efficiency, self-selecting CRISPRa enrichment strategy, which combined with piggyBac transposon technology enables rapid production of CRISPRa-ready cell populations compatible with a variety of downstream assays. We complement this with a new, optimized guide RNA scaffold that significantly enhances CRISPRa functionality. Finally, we describe a novel, synthetic guide RNA tool set that enables transient, population-wide gene activation when used with the self-selecting CRISPRa system. Taken together, this versatile platform greatly enhances the potential for CRISPRa across a wide variety of cellular contexts.

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“…In the extent of transcriptional activation achieved with CRISPRa, upregulation is dependent on the effect of native regulatory proteins as well as the native transcription level. When the CRISPRa system is correctly positioned, transcriptionally silent genes can be drastically upregulated, while enhanced activation of transcriptionally active genes is generally marginal (Strezoska et al, 2020). Problems of incorrectly positioned CRISPR guides could be potentially solved by deploying multiple spacers to the same promoter if the chosen CRISPRa/i system supports multiplexing.…”

Section: Crispr-based Transcriptional Regulationmentioning

confidence: 99%

Transcriptional Activation of Biosynthetic Gene Clusters in Filamentous Fungi

Mózsik

Iacovelli

Bovenberg

et al. 2022

Front. Bioeng. Biotechnol.

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Filamentous fungi are highly productive cell factories, many of which are industrial producers of enzymes, organic acids, and secondary metabolites. The increasing number of sequenced fungal genomes revealed a vast and unexplored biosynthetic potential in the form of transcriptionally silent secondary metabolite biosynthetic gene clusters (BGCs). Various strategies have been carried out to explore and mine this untapped source of bioactive molecules, and with the advent of synthetic biology, novel applications, and tools have been developed for filamentous fungi. Here we summarize approaches aiming for the expression of endogenous or exogenous natural product BGCs, including synthetic transcription factors, assembly of artificial transcription units, gene cluster refactoring, fungal shuttle vectors, and platform strains.

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CRISPR-mediated transcriptional activation with synthetic guide RNA

Cited by 16 publications

References 43 publications

Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi

Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi

A versatile, high-efficiency platform for CRISPR-based gene activation

Transcriptional Activation of Biosynthetic Gene Clusters in Filamentous Fungi

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