scholarly journals Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

Science ◽  
2021 ◽  
pp. eabe7106
Author(s):  
Chunlei Jiao ◽  
Sahil Sharma ◽  
Gaurav Dugar ◽  
Natalia L. Peeck ◽  
Thorsten Bischler ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Genetic Material ◽  
Simultaneous Detection ◽  
Type Ii ◽  
Rna Detection ◽  
Single Base ◽  
Dna Targeting ◽  
Single Base Resolution ◽  
Rna Complexes

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In Type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of “noncanonical” crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA-of-interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (Leveraging Engineered tracrRNAs and On-target DNAs for PArallel RNA Detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished SARS-CoV-2 and its D614G variant with single-base resolution in patient samples.

scholarly journals Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution

2019 ◽  
Vol 116 (14) ◽  
pp. 6784-6789 ◽  
Author(s):  
Vahid Khoddami ◽  
Archana Yerra ◽  
Timothy L. Mosbruger ◽  
Aaron M. Fleming ◽  
Cynthia J. Burrows ◽  
...  
Keyword(s):  
Ring Opening ◽  
Bisulfite Sequencing ◽  
Simultaneous Detection ◽  
Rna Modifications ◽  
Cdna Synthesis ◽  
Sequence Structure ◽  
Single Base ◽  
Single Base Resolution ◽  
Almost All ◽  
A Current

The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1–2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.

Mapping messenger RNA methylations at single base resolution

2021 ◽  
Vol 63 ◽  
pp. 28-37
Author(s):  
Jie Cao ◽  
Xiao Shu ◽  
Xin-Hua Feng ◽  
Jianzhao Liu
Keyword(s):  
Messenger Rna ◽  
Single Base ◽  
Single Base Resolution

scholarly journals Quantitative profiling of N6-methyladenosine at single-base resolution in stem-differentiating xylem of Populus trichocarpa using Nanopore direct RNA sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yubang Gao ◽  
Xuqing Liu ◽  
Bizhi Wu ◽  
Huihui Wang ◽  
Feihu Xi ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Populus Trichocarpa ◽  
Alternative Polyadenylation ◽  
High Accuracy ◽  
Single Base ◽  
Rna Immunoprecipitation ◽  
Single Base Resolution ◽  
Single Transcript ◽  
Quantitative Profiling ◽  
Identification And Quantification

AbstractThere are no comprehensive methods to identify N6-methyladenosine (m6A) at single-base resolution for every single transcript, which is necessary for the estimation of m6A abundance. We develop a new pipeline called Nanom6A for the identification and quantification of m6A modification at single-base resolution using Nanopore direct RNA sequencing based on an XGBoost model. We validate our method using methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and m6A-sensitive RNA-endoribonuclease–facilitated sequencing (m6A-REF-seq), confirming high accuracy. Using this method, we provide a transcriptome-wide quantification of m6A modification in stem-differentiating xylem and reveal that different alternative polyadenylation (APA) usage shows a different ratio of m6A.

Detection of EndogenousK-rasmRNA in Living Cells at a Single Base Resolution by a PNA Molecular Beacon

2012 ◽  
Vol 9 (3) ◽  
pp. 685-693 ◽  
Author(s):  
Yossi Kam ◽  
Abraham Rubinstein ◽  
Aviram Nissan ◽  
David Halle ◽  
Eylon Yavin
Keyword(s):  
Molecular Beacon ◽  
Living Cells ◽  
Single Base ◽  
Single Base Resolution

scholarly journals A single-base resolution map of an archaeal transcriptome

2009 ◽  
Vol 20 (1) ◽  
pp. 133-141 ◽  
Author(s):  
O. Wurtzel ◽  
R. Sapra ◽  
F. Chen ◽  
Y. Zhu ◽  
B. A. Simmons ◽  
...  
Keyword(s):  
Single Base ◽  
Single Base Resolution ◽  
Resolution Map

scholarly journals Gene specific-loci quantitative and single-base resolution analysis of 5-formylcytosine by compound-mediated polymerase chain reaction

2018 ◽  
Vol 9 (15) ◽  
pp. 3723-3728 ◽  
Author(s):  
Yafen Wang ◽  
Chaoxing Liu ◽  
Xiong Zhang ◽  
Wei Yang ◽  
Fan Wu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Demethylation ◽  
Chain Reaction ◽  
Single Base ◽  
Active Dna Demethylation ◽  
Key Players ◽  
Resolution Analysis ◽  
Single Base Resolution ◽  
Polymerase Chain

5-Formylcytosine (5fC) is known as one of the key players in the process of active DNA demethylation and displays essential epigenetic functions in mammals.

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