Sequencing 5-Formyluracil in Genomic DNA at Single-Base Resolution

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.

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Mapping messenger RNA methylations at single base resolution

Current Opinion in Chemical Biology ◽

10.1016/j.cbpa.2021.02.001 ◽

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

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

Genome Biology ◽

10.1186/s13059-020-02241-7 ◽

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.

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