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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. Truong et al. developed a cell-based reporter system, EXSISERS, that enables non-invasive quantification of the protein expression levels of exon-specific isoforms via intein-mediated protein splicing.

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. Exo-PE is an approach to improve prime editing efficacy for insertions while maintaining precision.

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. Truong et al. report a system to monitor RNA expression by modifying an intron within a gene of interest. This additional engineered transcript then hijacks nuclear export machinery for subsequent translation of a reporter gene.

Due to their long-term ecological stability, ancient lakes represent very interesting systems for studying differentiation and speciation processes. High degrees of endemism and specialisation are characteristic features of their fauna. The Malili lakes from the Indonesian island Sulawesi are considered an ancient limnic system, and recent research has increased the number of recognized species and the knowledge about ecological and morphological diversification within a variety of animal taxa. Here we show that the local endemic gecarcinucid freshwater crabs are more differentiated than qualitative morphological characters and the current taxonomy may indicate. The morphologically and ecologically well characterised species Nautilothelphusa zimmeri Balss, 1933 consists of two allopatric populations with one of the groups being more closely related to sympatric Parathelphusa ferruginea Chia and Ng, 2006 according to mitochondrial genetic data. One haplotype is even shared by both species. Nevertheless, in this study lack of gene flow between both species is demonstrated. To our knowledge, this is the first report of DNA sequence identity between two different genera along the same cytochrome oxidase I gene segment that is being proposed for barcoding studies.

Transcriptomics is a valuable technique for multiplexed monitoring of cellular states. However, its application has been predominantly limited to assays that necessitate cell fixation or lysis, which hinders real-time, longitudinal tracing of RNA expression in living cells. In this study, we present non-destructive transcriptomics by vesicular export (NTVE) to enable multi-time-point monitoring of RNA expression dynamics in live inducible reporter cell lines. Stabilized RNA reporter barcodes can be selectively packaged and exported from cells via virus-like particles (VLPs) containing selective affinity handles for convenient multichannel tracking of co-cultured cells. Using an engineered RNA adapter, NTVE exports cellular transcripts from inducible human and murine reporter cell lines with high concordance to conventional lysate-derived RNA-seq. NTVE allows monitoring of transcriptome changes in response to genetic and chemical perturbations in the same cells over time using standard sequencing workflows. NTVE can also be equipped with fusogens to deliver mRNA-encoded effectors or ribonucleoprotein gene editors from sender cells to activate gene reporters in co-cultured recipient cells. In addition, we demonstrate the utility of NTVE in monitoring hiPSC differentiation through daily NTVE analysis tracking multiple lineage-specific marker genes.Competing Interest StatementThe authors have declared no competing interest.

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Using specialized ice-nucleating proteins (INPs), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds, which may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INP-induced freezing have remained largely elusive. We investigate the structural basis for the interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae . Using vibrational sum-frequency generation (SFG) and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a β-helical structure in solution and at water surfaces. In this configuration, interaction between INPs and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with molecular-dynamics simulations and spectral calculations show that InaZ reorients at lower temperatures. This reorientation can enhance water interactions, and thereby the effectiveness of ice nucleation. Ice-nucleating proteins promote ice formation at high sub-zero temperatures, but the mechanism is still unclear. The authors investigate a model ice-nucleating protein at the air-water interface using vibrational sum frequency generation spectroscopy and simulations, revealing its reorientation at low temperatures, which increases contact with water molecules and promotes their ordering.

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Utilizing specialized ice-nucleating proteins (INPros), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds and may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INPro-induced freezing have remained largely elusive. In the present study, we investigated the folding and the structural basis for interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae strain R10.79. Using vibrational sum-frequency generation and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a β-helical structure in solution and at water surfaces. In this configuration, hydrogen bonding between INPros and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with spectral calculations and molecular-dynamics simulations shows that the INPro reorients at lower temperatures. We suggest that the reorientation can enhance order-inducing water interactions and, thereby, the effectiveness of ice nucleation by InaZ. Competing Interest Statement The authors have declared no competing interest.