RNA Modification & Editing

Small RNAs are modified with N-glycans and displayed on the surface of living cells

1-s2.0-S0092867421005031-main.pdf
Wed, 10/07/2020 - 00:17
Ryan A.Flynn, KayvonPedram, Stacy A.Malaker, Pedro J.Batista, Benjamin A.H.Smith, Alex G.Johnson, Benson M.George, KarimMajzoub, Peter W.Villalta, Jan E.Carette, Carolyn R.Bertozzi
Cell
https://www.sciencedirect.com/science/article/pii/S0092867421005031
UCSF HSW1057
Time
11:00am

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These “glycoRNAs” were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.

RNA Modification & Editing
RNA glycosylation
cell surface
small RNA
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StefanOberlin
Publication Title
Small RNAs are modified with N-glycans and displayed on the surface of living cells
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RNA_JC_012621_RNAglyco.pptx

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Search-and-replace genome editing without double-strand breaks or donor DNA

CRISPR-4.jpeg
Wed, 09/11/2019 - 00:00
Andrew V. Anzalone, Peyton B. Randolph, Jessie R. Davis, Alexander A. Sousa, Luke W. Koblan, Jonathan M. Levy, Peter J. Chen, Christopher Wilson, Gregory A. Newby, Aditya Raguram & David R. Liu
https://doi.org/10.1038/s41586-019-1711-4, 2019. Received: 26 August 2019; Accepted: 10 October 2019; Accelerated Article Preview Published online 21 October 2019.
https://www.nature.com/articles/s41586-019-1711-4_reference.pdf
Leonardo Ramos Ferreira
Time
12:00pm

Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2–5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutation without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing offers efficiency and product purity advantages over homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct about 89% of known pathogenic human genetic variants.

RNA Modification & Editing
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lcalviello
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Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts

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Sun, 09/01/2019 - 00:00
Campa CC, Weisbach NR1, Santinha AJ, Incarnato D, Platt RJ.
Nat Methods. 2019 Sep;16(9):887-893. doi: 10.1038/s41592-019-0508-6. Epub 2019 Aug 12.
https://www.nature.com/articles/s41592-019-0508-6.pdf
Gabriel Eades
Time
12:00pm

The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.

RNA Modification & Editing
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geades
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Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs

Fri, 11/23/2018 - 00:00
Liang Qu, Zongyi Yi, Shiyou Zhu, Chunhui Wang, Zhongzheng Cao, Zhuo Zhou, Pengfei Yuan, Ying Yu, Feng Tian, Zhiheng Liu, Ying Bao, Yanxia Zhao & Wensheng Wei
https://www.nature.com/articles/s41587-019-0178-z
Gabriel Eades
Time
12:00pm

Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity, delivery barrier or immunogenicity. Here, we present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change a specific adenosine to inosine. We show that arRNA, delivered by a plasmid or viral vector or as a synthetic oligonucleotide, achieves editing efficiencies of up to 80%. LEAPER is highly specific, with rare global off-targets and limited editing of non-target adenosines in the target region. It is active in a broad spectrum of cell types, including multiple human primary cell types, and can restore α-L-iduronidase catalytic activity in Hurler syndrome patient-derived primary fibroblasts without evoking innate immune responses. As a single-molecule system, LEAPER enables precise, efficient RNA editing with broad applicability for therapy and basic research.

RNA Modification & Editing
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geades
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RNA-guided DNA insertion with CRISPR-associated transposases

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Fri, 07/05/2019 - 00:00
Strecker J, Ladha A1, Gardner Z, Schmid-Burgk JL, Makarova KS, Koonin EV5, Zhang F6.
Science. 2019 Jul 5;365(6448):48-53. doi: 10.1126/science.aax9181. Epub 2019 Jun 6.
https://science.sciencemag.org/content/365/6448/48/tab-pdf
Olga Gulyaeva
Time
12:00pm

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

RNA Modification & Editing
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Olga Gulyaeva
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Updated Paper: "PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components"

mouse-4.jpg
Mon, 04/01/2019 - 00:00
Ma Z, Zhu P, Shi H, Guo L, Zhang Q, Chen Y1, Chen S, Zhang Z, Peng J, Chen J.
Nature. 2019 Apr;568(7751):259-263. doi: 10.1038/s41586-019-1057-y. Epub 2019 Apr 3.
https://www.nature.com/articles/s41586-019-1057-y.pdf
Ethan Caine
Time
12:00pm

The genetic compensation response (GCR) has recently been proposed as a possible explanation for the phenotypic discrepancies between gene-knockout and gene-knockdown1,2; however, the underlying molecular mechanism of the GCR remains uncharacterized. Here, using zebrafish knockdown and knockout models of the capn3a and nid1a genes, we show that mRNA bearing a premature termination codon (PTC) promptly triggers a GCR that involves Upf3a and components of the COMPASS complex. Unlike capn3a-knockdown embryos, which have small livers, and nid1a-knockdown embryos, which have short body lengths2, capn3a-null and nid1a-null mutants appear normal. These phenotypic differences have been attributed to the upregulation of other genes in the same families. By analysing six uniquely designed transgenes, we demonstrate that the GCR is dependent on both the presence of a PTC and the nucleotide sequence of the transgene mRNA, which is homologous to the compensatory endogenous genes. We show that upf3a (a member of the nonsense-mediated mRNA decay pathway) and components of the COMPASS complex including wdr5 function in GCR. Furthermore, we demonstrate that the GCR is accompanied by an enhancement of histone H3 Lys4 trimethylation (H3K4me3) at the transcription start site regions of the compensatory genes. These findings provide a potential mechanistic basis for the GCR, and may help lead to the development of therapeutic strategies that treat missense mutations associated with genetic disorders by either creating a PTC in the mutated gene or introducing a transgene containing a PTC to trigger a GCR.

RNA Modification & Editing
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Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9

cells-6.jpg
Thu, 05/02/2019 - 00:00
Shariati SA, Dominguez A, Xie S, Wernig M, Qi LS, Skotheim JM.
Mol Cell. 2019 May 2;74(3):622-633.e4. doi: 10.1016/j.molcel.2019.04.011.
Moritz Schlapansky
Time
12:00pm

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.

RNA Modification & Editing
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moritzs95
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Increasing the specificity of CRISPR systems with engineered RNA secondary structures

cell-2.jpeg
Mon, 04/15/2019 - 00:00
Kocak DD, Josephs EA, Bhandarkar V, Adkar SS, Kwon JB, Gersbach CA.
Nat Biotechnol. 2019 Apr 15. doi: 10.1038/s41587-019-0095-1. [Epub ahead of print]
Neil Tay
Time
12:00pm

CRISPR (clustered regularly interspaced short palindromic repeat) systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcus pyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems.

RNA Modification & Editing
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neiltay
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Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato

gene-reg-8.jpg
Wed, 01/16/2019 - 00:00
Tien Van Vu, Velu Sivankalyani, Eun-Jung Kim1, Mil Thi Tran1, Jihae Kim , Yeon Woo Sung, Duong Thi Hai Doan1, Jae-Yean Kim.
bioRxiv preprint first posted online Jan. 16, 2019; doi: http://dx.doi.org/10.1101/521419. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Yuhao Wang
Time
12:00pm


Genome editing via homology-directed repair (HDR) pathway in somatic plant cells was very inefficient compared to illegitimate repair by non-homologous end joining (NHEJ). Here, compared to a Cas9-based replicon system, we enhanced approximately 3-fold in the HDR-based genome editing efficiency via transient geminiviral replicon system equipping with CRISPR/LbCpf1 in tomato and obtained replicon-free, but with stable HDR alleles. Efficiency of CRISPR/LbCpf1-based HDR was significantly modulated by physical culture conditions such as temperature or light. A ten-day incubation at 31 o19 C under light/dark cycles after Agrobacterium-mediated transformation performed the best among conditions tested. Further, we developed multi-replicon system which is a novel tool to introduce effector components required for the increase of HDR efficiency. Even if it is still challenging, we also showed a feasibility of HDR-based genome editing without genomic integration of antibiotic marker or any phenotypic selection. Our work may pave a way for transgene-free rewriting of alleles of interest in asexually as well as
sexually reproducing plants.

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