scholarly journals Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells

2021 ◽  
Author(s):  
Alejandro Méndez-Mancilla ◽  
Hans-Hermann Wessels ◽  
Mateusz Legut ◽  
Anastasia Kadina ◽  
Megumi Mabuchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cd8 T Cells ◽  
Genetic Manipulation ◽  
Transient Gene Expression ◽  
Human Cells ◽  
Rna Modifications ◽  
Guide Rnas ◽  
Chemically Modified ◽  
Rna Targeting ◽  
Expression Modulation

RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to transiently modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be challenging and knockdown can be transient due to rapid crRNA degradation. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes enables transient gene expression modulation in primary CD4+ and CD8+ T-cells. This system represents a robust and efficient method to transiently modulate transcripts without genetic manipulation.

scholarly journals Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells

2021 ◽  
Author(s):  
Alejandro Méndez-Mancilla ◽  
Hans-Hermann Wessels ◽  
Mateusz Legut ◽  
Anastasia Kadina ◽  
Megumu Mabuchi ◽  
...  
Keyword(s):  
Human Cells ◽  
Guide Rnas ◽  
Chemically Modified

scholarly journals Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing

2018 ◽  
Author(s):  
Aamir Mir ◽  
Julia F. Alterman ◽  
Matthew R. Hassler ◽  
Alexandre J. Debacker ◽  
Edward Hudgens ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Genome Editing ◽  
Ex Vivo ◽  
Human Cells ◽  
Chemical Modifications ◽  
Oh Groups ◽  
Guide Rnas ◽  
Chemically Modified

RNA-based drugs depend on chemical modifications to increase potency and nuclease stability, and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. No studies have yet explored chemical modification at all positions of the crRNA guide and tracrRNA cofactor. Here, we have identified several heavily-modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2’-OH groups) that are functional in human cells. These designs demonstrate a significant breakthrough for Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.

scholarly journals Epitranscriptomic Signatures in lncRNAs and Their Possible Roles in Cancer

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 52 ◽  
Author(s):  
Sorina Dinescu ◽  
Simona Ignat ◽  
Andreea Lazar ◽  
Carolina Constantin ◽  
Monica Neagu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Modifications ◽  
Chemical Modifications ◽  
Chemically Modified ◽  
Additional Layer ◽  
Expression Control ◽  
Gene Expression Control ◽  
Level Of Knowledge ◽  
Non Coding Rnas ◽  
And Function

In contrast to the amazing exponential growth in knowledge related to long non-coding RNAs (lncRNAs) involved in cell homeostasis or dysregulated pathological states, little is known so far about the links between the chemical modifications occurring in lncRNAs and their function. Generally, ncRNAs are post-transcriptional regulators of gene expression, but RNA modifications occurring in lncRNAs generate an additional layer of gene expression control. Chemical modifications that have been reported in correlation with lncRNAs include m6A, m5C and pseudouridylation. Up to date, several chemically modified long non-coding transcripts have been identified and associated with different pathologies, including cancers. This review presents the current level of knowledge on the most studied cancer-related lncRNAs, such as the metastasis associated lung adenocarcinoma transcript 1 (MALAT1), the Hox transcript antisense intergenic RNA (HOTAIR), or the X-inactive specific transcript (XIST), as well as more recently discovered forms, and their potential roles in different types of cancer. Understanding how these RNA modifications occur, and the correlation between lncRNA changes in structure and function, may open up new therapeutic possibilities in cancer.

scholarly journals Engineering improved Cas13 effectors for targeted post-transcriptional regulation of gene expression

2021 ◽  
Author(s):  
Emeric J Charles ◽  
Shin Eui Kim ◽  
Gavin J Knott ◽  
Dylan Smock ◽  
Jennifer Doudna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Directed Evolution ◽  
Regulation Of Gene Expression ◽  
Exon Skipping ◽  
Cell Types ◽  
Human Cells ◽  
Translational Repression ◽  
E Coli ◽  
Rna Targeting

Cas13 is a family of unique RNA-targeting CRISPR-Cas effectors, making it an appealing tool for probing and perturbing RNA function. However only a few Cas13 homologs have been shown to mediate robust RNA targeting in human cells, suggesting that unknown elements may be limiting their efficacy. Furthermore, many Cas13 enzymes show high degrees of toxicity upon targeting and have not been shown to mediate specific knockdown in other cell types such as E. coli. Here, we show that catalytically inactive Cas13 enzymes can be repurposed for efficient translational repression in bacteria with no associated growth defects. To achieve this advance, we carried out a directed evolution screen to engineer functional Cas13a variants, and identified a number of stabilizing mutations, which enabled efficient post transcriptional knockdown of gene expression. In vitro characterization of the resulting engineered Lbu Cas13a mutant, termed eLbu, revealed both stabilization and altered cleavage kinetics. Finally, we show that eLbu can be used for efficient exon skipping in human cells. This work represents the first demonstration of targeted translational repression in E. coli using a CRISPR enzyme, as well as the first directed evolution of a Cas13a enzyme. Such a platform could allow for engineering other aspects of this protein family to obtain more robust RNA targeting tools.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

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