Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Li, Jiexin; Chen, Zhuojia; Chen, Feng; Ling, Yuyi; Peng, Yanxi; Luo, Nan; Wang, Hongsheng

doi:10.1101/614859

Cited by 4 publications

(1 citation statement)

References 44 publications

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“…More broadly, this programmable, small molecule-controllable RNA effector design provides a generalizable approach to study the RNA regulatory dynamics of an ever-growing list of known RNA regulatory proteins in live cells. Several recent advances in understanding the regulatory role of individual chemical modifications, such as m 6 A, have relied on programmable site-specific targeting technology such as RNA-targeting Cas9 (RCas9) or Cas13. − For example, a programmable dCas13b-FTO protein was instrumental in demonstrating the role m 6 A modifications on specific chromosome-associated regulatory RNAs play in transcription . Despite these advances in understanding regulatory roles of m 6 A on specific transcripts, the temporal component of their regulation has yet to be elucidated.…”

Section: Discussionsupporting

confidence: 61%

Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation

2020

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All aspects of mRNA lifetime and function, including its stability, translation into protein, and trafficking through the cell, are tightly regulated through coordinated post-transcriptional modifications and interactions with a multitude of RNA effector proteins. Despite the increasing recognition of RNA regulation as a critical layer of mammalian gene expression control and its increasing excitement as a therapeutic target, tools to study and control RNA regulatory mechanisms with temporal precision in their endogenous environment are lacking. Here, we present small molecule-inducible RNA-targeting effectors based on our previously developed CRISPR/Cas-inspired RNA targeting system (CIRTS). The CIRTS biosensor platform is based on guide RNA (gRNA)-dependent RNA binding domains that interact with a target transcript using Watson–Crick–Franklin base pair interactions. Addition of a small molecule recruits an RNA effector to the target transcript, thereby eliciting a local effect on the transcript. In this work, we showcase that these CIRTS biosensors can trigger inducible RNA editing, degradation, or translation on target transcripts in a small molecule-dependent manner. We further go on to show that the CIRTS RNA base editor biosensor can induce RNA base editing in a small molecule-controllable manner in vivo . Collectively this work provides a new set of tools to probe the dynamics of RNA regulatory systems and control gene expression at the RNA level.

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Section: Discussionsupporting

confidence: 61%

Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation

2020

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m6A mRNA Modification as a New Layer of Gene Regulation in Plants

et al. 2020

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Massively parallel Cas13 screens reveal principles for guide RNA design

et al. 2020

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Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knock-down without altering the genome. To define rules for the design of Cas13d guide RNAs, we conducted massively-parallel screens targeting mRNAs of a green fluorescent protein (GFP) transgene and CD46, CD55 and CD71 cell surface proteins in human cells. In total, we measured the activity of 24,460 guide RNAs with and without mismatches relative to the target sequences. Knock-down efficacy is driven by guide RNA-specific features and target site context. Single mismatches generally reduce knock-down to a modest degree, but spacer nucleotides 15 – 21 are largely intolerant to target site mismatches. We developed a computational model to identify optimal guide RNAs and confirm its generalizability testing 3,979 guides targeting mRNAs of 48 endogenous genes. We show that Cas13 can be used in forward transcriptomic pooled screens and, using our model, predict optimized Cas13 guide RNAs for all protein-coding transcripts in the human genome.

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Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Cited by 4 publications

References 44 publications

Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation

Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation

m6A mRNA Modification as a New Layer of Gene Regulation in Plants

Massively parallel Cas13 screens reveal principles for guide RNA design

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