Exonuclease-enhanced prime editors.

Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.

Identification of ocular regulatory functions of core histone variant H3.2.

The posttranscriptional modifications (PTM) of the Histone H3 family play an important role in ocular system differentiation. However, there has been no study on the nature of specific Histone H3 subtype carrying these modifications. Fortuitously, we had previously identified a dominant small-eye mutant Aey69 mouse with a mutation in the H3.2 encoding Hist2h3c1 gene (Vetrivel et al., 2019). In continuation, in the present study, the role of Histone H3.2 with relation to the microphtalmic Aey69 has been elaborated. Foremost, a transgenic mouse line expressing the fusion protein H3.2-GFP was generated using Crispr/Cas9. The approach was intended to confer a unique tag to the Hist2h3c1 gene which is similar in sequence and encoded protein structure to other histones. The GFP tag was then used for ChIP Seq analysis of the genes regulated by H3.2. The approach revealed ocular specific H3.2 targets including Ephrin family genes. Altered enrichment of H3.2 was found in the mutant Aey69 mouse, specifically around the ligand Efna5 and the receptor Ephb2. The effect of this altered enrichment on Ephrin signaling was further analysed by QPCR and immunohistochemistry. This study identifies Hist2h3c1 encoded H3.2 as an important epigenetic player in ocular development. By binding to specific regions of ocular developmental factors Histone H3.2 facilitates the function of these genes for successful early ocular development.

Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs.

Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.

Parkinson's disease motor symptoms rescue by CRISPRa-reprogramming astrocytes into GABAergic neurons.

Direct reprogramming based on genetic factors resembles a promising strategy to replace lost cells in degenerative diseases such as Parkinson's disease. For this, we developed a knock-in mouse line carrying a dual dCas9 transactivator system (dCAM) allowing the conditional in vivo activation of endogenous genes. To enable a translational application, we additionally established an AAV-based strategy carrying intein-split-dCas9 in combination with activators (AAV-dCAS). Both approaches were successful in reprogramming striatal astrocytes into induced GABAergic neurons confirmed by single-cell transcriptome analysis of reprogrammed neurons in vivo. These GABAergic neurons functionally integrate into striatal circuits, alleviating voluntary motor behavior aspects in a 6-OHDA Parkinson's disease model. Our results suggest a novel intervention strategy beyond the restoration of dopamine levels. Thus, the AAV-dCAS approach might enable an alternative route for clinical therapies of Parkinson's disease.

Non-invasive and high-throughput interrogation of exon-specific isoform expression.

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

Nucleus size and DNA accessibility are linked to the regulation of paraspeckle formation in cellular differentiation.

BACKGROUND: Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. RESULTS: As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1, are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. CONCLUSIONS: Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility.

Development of an intein-mediated split-Cas9 system for gene therapy.

Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.

Design and characterization of a modular membrane protein anchor to functionalize the moss Physcomitrella patens with extracellular catalytic and/or binding activities.

Heterologous enzymes and binding proteins were secreted by the moss Physcomitrella patens or anchored extracellularly on its cell membrane in order to functionalize the apoplast as a biochemical reaction compartment. This modular membrane anchoring system utilizes the signal peptide and the transmembrane segment of the somatic embryogenesis receptor-like kinase (SERK), which were identified in a comprehensive bioinformatic analysis of the P. patens genome. By fusing the soluble enzyme NanoLuc luciferase to the signal peptide, its secretion capability was confirmed in vivo. The membrane localization of hybrid proteins comprising the SERK signal peptide, NanoLuc or other functional modules, the SERK transmembrane anchor, and a C-terminal GFP reporter was demonstrated using fluorescence microscopy as well as site-specific proteolytic release of the extracellular enzyme domain. Our membrane anchoring system enables the expression of various functional proteins in the apoplast of P. patens, empowering this photoautotrophic organism for biotechnological applications.

Cardiac myocyte-secreted cAMP exerts paracrine action via adenosine receptor activation.

Acute stimulation of cardiac ß-adrenoceptors is crucial to increasing cardiac function under stress; however, sustained ß-adrenergic stimulation has been implicated in pathological myocardial remodeling and heart failure. Here, we have demonstrated that export of cAMP from cardiac myocytes is an intrinsic cardioprotective mechanism in response to cardiac stress. We report that infusion of cAMP into mice averted myocardial hypertrophy and fibrosis in a disease model of cardiac pressure overload. The protective effect of exogenous cAMP required adenosine receptor signaling. This observation led to the identification of a potent paracrine mechanism that is dependent on secreted cAMP. Specifically, FRET-based imaging of cAMP formation in primary cells and in myocardial tissue from murine hearts revealed that cardiomyocytes depend on the transporter ABCC4 to export cAMP as an extracellular signal. Extracellular cAMP, through its metabolite adenosine, reduced cardiomyocyte cAMP formation and hypertrophy by activating A1 adenosine receptors while delivering an antifibrotic signal to cardiac fibroblasts by A2 adenosine receptor activation. Together, our data reveal a paracrine role for secreted cAMP in intercellular signaling in the myocardium, and we postulate that secreted cAMP may also constitute an important signal in other tissues.