PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.

TOBF1 modulates mouse embryonic stem cell fate through regulating alternative splicing of pluripotency genes.

Embryonic stem cells (ESCs) can undergo lineage-specific differentiation, giving rise to different cell types that constitute an organism. Although roles of transcription factors and chromatin modifiers in these cells have been described, how the alternative splicing (AS) machinery regulates their expression has not been sufficiently explored. Here, we show that the long non-coding RNA (lncRNA)-associated protein TOBF1 modulates the AS of transcripts necessary for maintaining stem cell identity in mouse ESCs.

Identification of G-quadruplex structures in MALAT1 lncRNA that interact with nucleolin and nucleophosmin.

Nuclear-retained long non-coding RNAs (lncRNAs) including MALAT1 have emerged as critical regulators of many molecular processes including transcription, alternative splicing and chromatin organization. Here, we report the presence of three conserved and thermodynamically stable RNA G-quadruplexes (rG4s) located in the 3' region of MALAT1. Using rG4 domain-specific RNA pull-down followed by mass spectrometry and RNA immunoprecipitation, we demonstrated that the MALAT1 rG4 structures are specifically bound by two nucleolar proteins, Nucleolin (NCL) and Nucleophosmin (NPM).

Fine-tuning miR-21 expression and inhibition of EMT in breast cancer cells using aromatic-neomycin derivatives.

MicroRNAs (miRs) are a class of endogenously expressed non-coding RNAs that negatively regulate gene expression within cells and participate in maintaining cellular homeostasis. By targeting 3' UTRs of target genes, individual miRs can control a wide array of gene expressions. Previous research has shed light upon the fact that aberrantly expressed miRs within cells can pertain to diseased conditions, such as cancer.

Alternative splicing modulation mediated by G-quadruplex structures in MALAT1 lncRNA.

MALAT1, an abundant lncRNA specifically localized to nuclear speckles, regulates alternative-splicing (AS). The molecular basis of its role in AS remains poorly understood. Here, we report three conserved, thermodynamically stable, parallel RNA-G-quadruplexes (rG4s) present in the 3' region of MALAT1 which regulates this function.

Diagnostic and Prognostic Potential of MiR-379/656 MicroRNA Cluster in Molecular Subtypes of Breast Cancer.

Introduction: Breast cancer is the most frequently diagnosed cancer globally and is one of the most important contributors to cancer-related deaths. Earlier diagnosis is known to reduce mortality, and better biomarkers are needed. MiRNA clusters often co-express and target mRNAs in a coordinated fashion, perturbing entire pathways; they thus merit further exploration for diagnostic or prognostic use.

Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules.

Human Brain Shows Recurrent Non-Canonical MicroRNA Editing Events Enriched for Seed Sequence with Possible Functional Consequence.

RNA editing is a post-transcriptional modification, which can provide tissue-specific functions not encoded in DNA. Adenosine-to-inosine is the predominant editing event and, along with cytosine-to-uracil changes, constitutes canonical editing. The rest is non-canonical editing.

Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing.

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci.