Mettl14-mediated m6A modification ensures the cell-cycle progression of late-born retinal progenitor cells.

Neural progenitor cells lengthen their cell cycle to prime themselves for differentiation as development proceeds. It is currently not clear how they counter this lengthening and avoid being halted in the cell cycle. We show that N6-methyladenosine (m6A) methylation of cell-cycle-related mRNAs ensures the proper cell-cycle progression of late-born retinal progenitor cells (RPCs), which are born toward the end of retinogenesis and have long cell-cycle length. Conditional deletion of Mettl14, which is required for depositing m6A, led to delayed cell-cycle exit of late-born RPCs but has no effect on retinal development prior to birth. m6A sequencing and single-cell transcriptomics revealed that mRNAs involved in elongating the cell cycle were highly enriched for m6A, which could target them for degradation and guarantee proper cell-cycle progression. In addition, we identified Zfp292 as a target of m6A and potent inhibitor of RPC cell-cycle progression.

Multiplexed genome regulation in vivo with hyper-efficient Cas12a.

Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.

Identification of cis-regulatory modules for adeno-associated virus-based cell-type-specific targeting in the retina and brain.

Adeno-associated viruses (AAVs) targeting specific cell types are powerful tools for studying distinct cell types in the central nervous system (CNS). Cis-regulatory modules (CRMs), e.g., enhancers, are highly cell-type-specific and can be integrated into AAVs to render cell type specificity. Chromatin accessibility has been commonly used to nominate CRMs, which have then been incorporated into AAVs and tested for cell type specificity in the CNS. However, chromatin accessibility data alone cannot accurately annotate active CRMs, as many chromatin-accessible CRMs are not active and fail to drive gene expression in vivo. Using available large-scale datasets on chromatin accessibility, such as those published by the ENCODE project, here we explored strategies to increase efficiency in identifying active CRMs for AAV-based cell-type-specific labeling and manipulation. We found that prescreening of chromatin-accessible putative CRMs based on the density of cell-type-specific transcription factor binding sites (TFBSs) can significantly increase efficiency in identifying active CRMs. In addition, generation of synthetic CRMs by stitching chromatin-accessible regions flanking cell-type-specific genes can render cell type specificity in many cases. Using these straightforward strategies, we generated AAVs that can target the extensively studied interneuron and glial cell types in the retina and brain. Both strategies utilize available genomic datasets and can be employed to generate AAVs targeting specific cell types in CNS without conducting comprehensive screening and sequencing experiments, making a step forward in cell-type-specific research.

Cell type- and stage-specific expression of Otx2 is regulated by multiple transcription factors and cis-regulatory modules in the retina.

Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is necessary. We analyzed how the expression of the homeobox TF, orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the mouse retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs, respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages. We conclude that retinal cells use a cohort of TFs with different expression patterns and multiple CRMs with different chromatin configurations to regulate the expression of Otx2 precisely.