Biology subjects

Koirala, A.

Publications and source records attributed to Koirala, A..

4 recordsLinked to original sources

bioRxiv2025

eSPLIT and iSWAP: CRISPR-Mediated Conditional Allele Engineering with Short Artificial Introns

Engineering conditional alleles remains a major challenge in functional genomics. Short Artificial Introns (SAIs) have emerged as powerful tools to simplify allele design and characterization, yet practical guidelines for their implementation remain limited. Here, we describe two streamlined strategies, eSPLIT (exon split) and iSWAP (intron swap), that enable efficient generation of conditional alleles using SAIs. In eSPLIT, a compact cassette containing essential intronic elements flanked by loxP sites is integrated within an exon, whereas in iSWAP, a native intron is replaced with an SAI. In the absence of Cre recombinase, the SAI is recognized as an intron and removed by the splicing machinery, allowing normal gene expression. Following Cre-mediated recombination, excision of critical intronic sequences disrupts splicing, leaving residual sequences, including stop codons in all reading frames, thereby causing premature translation termination and gene inactivation. We optimized these approaches by generating a series 18 SCYL1 alleles in human cells, validated their general applicability in 5 mouse models across multiple genes and further extended the approach to the FLP-FRT system in vivo. By defining practical rules for SAI placement, we establish a robust and scalable framework for engineering conditional alleles, with broad utility in functional genomics and disease modeling. SHORT 100 - WORD ABSTRACTWe introduce eSPLIT and iSWAP, two streamlined strategies for generating conditional alleles using a short artificial intron (SAI). eSPLIT integrates a loxP-flanked SAI within an exon, while iSWAP replaces a native intron. Cre-mediated excision disrupts SAI splicing, leading to gene inactivation. Optimized using SCYL1 alleles in human cells and validated in mouse models across multiple genes, these strategies provide a simple and efficient approach for engineering conditional alleles. By outlining design rules for SAI placement, we provide a robust and scalable strategy for functional genomics and disease modeling.

genomics↗

bioRxiv2025

Cell type-independent timekeeping gene modules enable embryonic stage prediction in zebrafish

Gene expression changes across embryonic development reflect both differentiation and genes whose expression varies strictly with developmental time, independent of cell type. Multiple embryonic timing systems set the onset and pace of developmental events, and blocking transcription arrests many of these events. However, the genes comprising the vertebrate embryonic timing system(s) remain largely unknown. To identify genes whose expression changes with time alone, we examine naive zebrafish embryonic explants that form only two tissue types yet maintain developmental timing, thus uncoupling developmental age from most differentiation programs. By comparing longitudinal gene expression in naive explants with Nodal-induced explants that differentiate into all three germ layers, we identify "timekeeping" genes whose temporal expression patterns vary strictly with developmental age. Consensus clustering of temporally dynamic genes identified 20 gene clusters, termed "chrono-constitutive modules" (CCMs), that maintain distinct schedules of expression regardless of tissue type. These CCM trajectories are similar in intact zebrafish embryos and single embryonic cells of multiple distinct lineages. Enrichment analysis of microRNA targets and transcription factor regulons within the CCMs further reveal distinct putative regulators of several modules. Strikingly, CCM expression patterns are also largely conserved during early development of another fish species, Japanese medaka. Machine learning models trained on only zebrafish CCM transcript levels accurately predict the developmental age of embryonic explants, intact embryos, and even individual embryonic cells, demonstrating their utility in developmental timekeeping. These results support the existence of transcriptional timekeeping during early development and demonstrate its utility in embryonic stage prediction.

developmental biology↗

bioRxiv2025

Covalent Inhibition of the Human Papillomavirus Type 16 E6 Protein Restores p53 and Suppresses HPV-Driven Tumorigenesis

High-risk human papillomavirus (HPV) infections are the etiology of approximately 5% of all cancers worldwide, including cervical, anal, and oropharyngeal malignancies. HPV E6 is a multifunctional oncoprotein that drives tumorigenesis and is best known for bridging the ubiquitin ligase E6AP (UBE3A) and p53 into a complex that leads to proteasome mediated destruction of p53. We developed small molecule inhibitors that covalently bind to cysteine-51 (Cys-51) in HPV16 E6. In HPV16-positive cancer cells, these compounds increase p53 protein levels and activate p53-dependent transcriptional programs associated with apoptosis and senescence, resulting in reduced tumor cell viability. In vivo, E6 inhibition suppresses the growth of human HPV16 expressing cervical and oropharyngeal tumor cell lines in mice. The strategy of targeting a viral oncoprotein with a covalent inhibitor demonstrates a genotype-specific therapeutic strategy for HPV-associated cancers and premalignant infections, addressing a significant unmet need in current treatment options.

cancer biology↗

bioRxiv2022

Clonal dynamics of SARS-CoV-2-specific T cells in children and adults with COVID-19

Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV- 2-specific naive T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. More naive interferon-activated CD4+ T cells were recruited into the memory compartment and recovery was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection. HIGHLIGHTSO_LIChildren have diverse polyclonal SARS-CoV-2-specific naive T cells C_LIO_LIAdults have clonally expanded exhausted SARS-CoV-2-specific memory T cells C_LIO_LIInterferon-activated naive T cells differentiate into memory T cells in adults but not children C_LIO_LIAdults but not children develop robust memory T cell responses to SARS-CoV-2 C_LI O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/478400v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@e9586org.highwire.dtl.DTLVardef@17aaf37org.highwire.dtl.DTLVardef@18575e0org.highwire.dtl.DTLVardef@fde4ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗