Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

Chen, Yuxi; Liu, Jiaqi; Zhi, Shengyao; Zheng, Qi; Ma, Wenbin; Huang, Junjiu; Liu, Yizhi; Liú, Dan; Liang, Puping; Songyang, Zhou

doi:10.1038/s41467-020-16880-8

Cited by 47 publications

(56 citation statements)

References 65 publications

(102 reference statements)

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“…For instance, VPR fusions to Cascade systems from Pseudomonas aeruginosa (PaeCascade-VPR) guided by 32 nt crRNAs are capable of activating gene transcription of therapeutic genes such as HBB and HBG to treat β-thalassemia, without predicted off-target activities. This recent technology is very sensitive to crRNA-DNA mismatches, including those very distal to the PAM sequence ( 234 ). Despite this high specificity, the delivery in vivo of such bulky multi-modular complexes represent a potential obstacle for subsequent translational applications.…”

Section: Specificity Of Epigenome Editing Toolsmentioning

confidence: 99%

Epigenome engineering: new technologies for precision medicine

Sgro

Blancafort

2020

Nucleic Acids Research

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Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more ‘normal-like state’, having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.

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Section: Specificity Of Epigenome Editing Toolsmentioning

confidence: 99%

Epigenome engineering: new technologies for precision medicine

Sgro

Blancafort

2020

Nucleic Acids Research

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“…In prokaryotes, repurposing of endogenous CRISPR–Cas systems to genome editing or gene regulation has been reported ( 25 , 30 ). In eukaryotic cells, type I-E and type I-F systems have been utilized to regulate gene expression by fusing transcriptional activators/repressors to Cas proteins in plant ( 29 ) and human ( 31 ) cells, respectively. The type I-E system has also been applied to genome editing.…”

Section: Introductionmentioning

confidence: 99%

Genome editing in mammalian cells using the CRISPR type I-D nuclease

Osakabe

Wada

Murakami

et al. 2021

Nucleic Acids Research

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Adoption of CRISPR–Cas systems, such as CRISPR–Cas9 and CRISPR–Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense (‘Cascade’: Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR–Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR–Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR–Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

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“…Although we merely illustrated gene activation with large dynamic ranges by controlling the cleavage of coding sequences, we anticipate the great potential of cognate acrRNA and pre-crRNAs coupling as riboregulators in the future, e.g., by gene activation through RBS exposure 21 , or induction of circular RNA formation 20 . Significantly, design of acrRNAs may enable accurate control of type I CRISPR for genome editing 43 or transcriptional controls 44 . While acr proteins 34, 35 can only bind to Cas proteins but not distinguish specific crRNAs or guide RNAs, programmable anti-CRISPR RNAs (acrRNAs) may enable crRNA-specific repression, supplementing the anti-CRISPR toolkit.…”

Section: Discussionmentioning

confidence: 99%

Anti-CRISPR RNAs: designing universal riboregulators with deep learning of Csy4-mediated RNA processing

Guo

Song

Lindner

2020

Preprint

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RNA-based regulation offers a promising alternative of protein-based transcriptional networks. However, designing synthetic riboregulators with desirable functionalities using arbitrary sequences remains challenging, due in part to insufficient exploration of RNA sequence-to-function landscapes. Here we report that CRISPR-Csy4 mediates a nearly all-or-none processing of precursor CRISPR RNAs (pre-crRNAs), by profiling Csy4 binding sites flanked by > 1 million random sequences. This represents an ideal sequence-to-function space for universal riboregulator designs. Lacking discernible sequence-structural commonality among processable pre-crRNAs, we trained a neural network for accurate classification (f1-score ≈ 0.93). Inspired by exhaustive probing of palindromic flanking sequences, we designed anti-CRISPR RNAs (acrRNAs) that suppress processing of pre-crRNAs via stem stacking. We validated machine-learning-guided designs with >30 functional pairs of acrRNAs and pre-crRNAs to achieve switch-like properties. This opens a wide range of plug-and-play applications tailored through pre-crRNA designs, and represents a programmable alternative to protein-based anti-CRISPRs.

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Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

Cited by 47 publications

References 65 publications

Epigenome engineering: new technologies for precision medicine

Epigenome engineering: new technologies for precision medicine

Genome editing in mammalian cells using the CRISPR type I-D nuclease

Anti-CRISPR RNAs: designing universal riboregulators with deep learning of Csy4-mediated RNA processing

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