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.

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.

A-to-I editing in human miRNAs is enriched in seed sequence, influenced by sequence contexts and significantly hypoedited in glioblastoma multiforme.

Editing in microRNAs, particularly in seed can significantly alter the choice of their target genes. We show that out of 13 different human tissues, different regions of brain showed higher adenosine to inosine (A-to-I) editing in mature miRNAs. These events were enriched in seed sequence (73.

Altered expression and editing of miRNA-100 regulates iTreg differentiation.

RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4(+) T cell differentiation. CD4(+) T cells are an integral component of the adaptive immune system.

Genome-wide analysis reveals downregulation of miR-379/miR-656 cluster in human cancers.

Background: MicroRNAs (miRNAs) are non-uniformly distributed in genomes and ~30% of the miRNAs in the human genome are clustered. In this study we have focused on the imprinted miRNA cluster miR-379/miR-656 on 14q32.31 (hereafter C14) to test their coordinated function.