Mourelatos, H. | Search bioRxiv

bioRxiv2022

Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2

We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly gRNA/library design in custom genomes. GuideScan2 analysis identified widespread confounding effects of low-specificity gRNAs in published CRISPR knockout, interference and activation screens and enabled construction of a ready-to-use gRNA library that reduced off-target effects in a novel gene essentiality screen. GuideScan2 also enabled the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome.

genomics↗

bioRxiv2020

Post-Transcriptional Regulation of Antiviral Gene Expression by N6-Methyladenosine

Type I interferons (IFN) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. These ISGs require strict regulation for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a new role for the RNA base modification N6-methyladenosine (m6A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m6A-immunoprecipitation and sequencing, we identified a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determined that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A binding-dependent manner. Importantly, we found that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as a post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction.

immunology↗